Frog Skeletal Muscle Fibers Research Articles

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1H1630 トロポニンを除去したカエル骨格筋スキンドファイバーからのX線回折

Force-velocity and unloaded shortening velocity during graded potassium contractures in frog skeletal muscle fibres.

Steady-state conditions of contraction, at maximal and submaximal forces, were produced in intact single muscle fibres, from Rana esculenta, using full tetani and graded K+-contractures. The uniformity in radial direction. of spreading of activation produced in K+-contractures, was checked in relation to the fibre diameters. The absolute isometric force was similar in tetani and maximal contractures, for fibres with diameters between 40 and 60 microm, but not for fibres with diameters greater than about 70 microm in which contracture force never reached tetanic force. The force [K+]o relation was similar for fibres with diameters between 40 and 60 microm. but it was right shifted and it had a minor slope for fibres with diameters greater than 65-70 microm. This suggests that only in the small diameter fibres (40-60 microm) the activation does not fail to penetrate uniformly from the surface towards the fibre core. For fibres selected in the diameter range between 40 and 60 microm, force-velocity relations and unloaded shortening velocities were determined in tetani and maximal and submaximal contractures. Data were obtained across a force range of 0.3 to 1 P0 (tetanic plateau force). Controlled velocity method was used to obtain force-velocity relations, and slack test to determine the unloaded shortening velocity (VU). The values of the parameters characterising the force velocity relation (V0 and a/P0) and VU as determined by the slack test did not differ significantly in tetani and contractures, independent of the activation level or absolute force developed by the fibre. These results show that. at least within the range of forces tested. crossbridge kinetics is independent of the number of cycling crossbridges, in agreement with the prediction of the 'recruitment' model of myofilament activation.

Hydrogen peroxide decelerates recovery of action potential after high-frequency fatigue in skeletal muscle.

Effects of reactive oxygen species (ROS), especially hydrogen peroxide (H(2)O(2)), on recovery of action potential by resting for 30 min after high-frequency fatigue were studied using frog skeletal muscle fibers. After stimulation at a frequency of 50 HZ for 2 min, the action potential amplitude was decreased by 14.5 mV from controls, and resting membrane was depolarized by 15.4 mV. Action potential duration was also prolonged by high-frequency stimulation (1.5 ms in controls to 2.6 ms). The high-frequency stimulation used here caused no muscle damage. The action potential was partially improved after a 30-min rest. Addition of catalase at 500 units/ml or H(2)O(2) at 0.5 mM to sartorius muscle did not alter any of the parameters of the action potential after high-frequency stimulation. Treatment with catalase accelerated post-fatigue recovery of the action potential. Application of H(2)O(2) delayed post-fatigue recovery of resting and action potentials. When added to detubulated toe muscle fibers, catalase no longer improved the attenuation of action potential induced by high-frequency stimulation, even after a 30-min rest. These findings suggest that removal of H(2)O(2) from transverse tubules is effective for post-fatigue recovery of action potential in skeletal muscle.

Frog skeletal muscle fibers recovering from fatigue have reduced charge movement.

Following prolonged exercise, muscle force production is often impaired. One possible cause of this force deficit is impaired intracellular activation. We have used single skeletal muscle fibers from the lumbrical muscle of Xenopus laevis to study the effects of fatigue on excitation-contraction coupling. Fatigue was induced in 13 intact fibers. Five fibers recovered in normal Ringer only (fatigued-only fibers). The remaining eight fibers were subjected to a brief hypotonic treatment (F-H fibers) that is known to prolong the effects of fatigue. Intramembrane charge movement, changes in intracellular calcium concentration ([Ca2+]i) and force transients were measured in a single Vaseline gap chamber under voltage clamp. In F-H fibers, membrane capacitance was reduced. Confocal microscopy showed that this was not due to closure of the transverse tubules. The amount of normalized intramembrane charge was reduced from 21.0 +/- 2.8 nC/microF (n = 10) in rested fibers to 12.2 +/- 1.1 nC/microF in F-H fibers. However, the voltage dependence of intramembrane charge movement was unchanged. In F-H fibers, force production was virtually abolished. This was the consequence of the greatly reduced [Ca2+]i accompanying a depolarizing pulse. In recovering fatigued-only fibers, while the maximal available charge was not significantly smaller (18.3 +/- 1.1 nC/ microF), both calcium and force were reduced, albeit to a lesser extent than in F-H fibers. The data are consistent with a model where fatigue reduces the number of voltage sensors in the t-tubules and, in addition, alters the coupling between the remaining functional voltage sensors and the calcium channels of the sarcoplasmic reticulum.

Structural responses to the photolytic release of ATP in frog muscle fibres, observed by time-resolved X-ray diffraction.

1. Structural changes following the photolytic release of ATP were observed in single, permeabilised fibres of frog skeletal muscle at 5-6 C, using time-resolved, low-angle X-ray diffraction. The structural order in the fibres and their isometric function were preserved by cross-linking 10-20 % of the myosin cross-bridges to the thin filaments. 2. The time courses of the change in force, stiffness and in intensity of the main equatorial reflections (1,0) and (1,1), of the third myosin layer line (M3) at a reciprocal spacing of (14.5 nm)-1 on the meridian and of the first myosin-actin layer line (LL1) were measured with 1 ms time resolution. 3. In the absence of Ca2+, photolytic release of ATP in muscle fibres initially in the rigor state caused the force and stiffness to decrease monotonically towards their values in relaxed muscle fibres. 4. In the presence of Ca2+, photolytic release of ATP resulted in an initial rapid decrease in force, followed by a slower increase to the isometric plateau. Muscle fibre stiffness decreased rapidly to approximately 65 % of its value in rigor. 5. In the absence of Ca2+, changes on the equator, in LL1 and in M3 occurred with a time scale comparable to that of the changes in tension and stiffness. 6. In the presence of Ca2+, the changes on the equator and LL1 occurred simultaneously with the early phase of tension decrease. The changes in the intensity of M3 (IM3) occurred on the time scale of the subsequent increase in force. The time courses of the changes in tension and IM3 were similar following the photolytic release of 0. 33 or 1.1 mM ATP. However the gradual return towards the rigor state began earlier when only 0.33 mM ATP was released. 7. In the presence of Ca2+, the time course of changes in IM3 closely mimicked that of force development following photolytic release of ATP. This is consistent with models that propose that force development results from a change in the average orientation of cross-bridges, although other factors, such as their redistribution, may also be involved.

Na(+)-Ca2+ exchange induces low Na+ contracture in frog skeletal muscle fibers after partial inhibition of sarcoplasmic reticulum Ca(2+)-ATPase.

Contractile responses due to reduction in external sodium concentration ([Na+]o) were investigated in twitch skeletal muscle fibers of frog semitendinosus. Experiments were conducted after partial inhibition of sarcoplasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid (CPA). In the absence of CPA, Na+ withdrawal failed to produce any change in resting tension. In the presence of CPA (2-10 microM), [Na+]o reduction induced a transient contracture without a significant change in the resting membrane potential. The amplitude of the contracture displayed a step dependence on [Na+]o, was increased by K(+)-free medium and was prevented in Ca(2+)-free medium. This contracture was inhibited by various blockers of the Na(+)-Ca2+ exchange but was little affected by inhibitors of sarcolemmal Ca(2+)-ATPase or mitochondria. When sarcoplasmic reticulum function was impaired, low-Na+ solutions caused no contracture. These results provide evidence that skeletal muscle fibers possess a functional Na(+)-Ca2+ exchange which can mediate sufficient Ca2+ entry to activate contraction by triggering Ca2+ release from sarcoplasmic reticulum when the sodium electrochemical gradient is reduced, and sarcoplasmic reticulum Ca(2+)-ATPase is partially inhibited. This indicates that when the sarcoplasmic reticulum Ca(2+)-ATPase is working (no CPA), Ca2+ fluxes produced by the exchanger are buffered by the sarcoplasmic reticulum. Thus the Na(+)-Ca2+ exchange may be one of the factors determining sarcoplasmic reticulum Ca2+ content and thence the magnitude of the release of Ca2+ from the sarcoplasmic reticulum.

Na + -Ca 2+ exchange induces low Na + contracture in frog skeletal muscle fibers after partial inhibition of sarcoplasmic reticulum Ca 2+ -ATPase

Na + -Ca 2+ exchange induces low Na + contracture in frog skeletal muscle fibers after partial inhibition of sarcoplasmic reticulum Ca 2+ -ATPase

Calcium release flux underlying Ca2+ sparks of frog skeletal muscle.

An algorithm for the calculation of Ca2+ release flux underlying Ca2+ sparks (Blatter, L.A., J. Hüser, and E. Ríos. 1997. Proc. Natl. Acad. Sci. USA. 94:4176-4181) was modified and applied to sparks obtained by confocal microscopy in single frog skeletal muscle fibers, which were voltage clamped in a two-Vaseline gap chamber or permeabilized and immersed in fluo-3-containing internal solution. The performance of the algorithm was characterized on sparks obtained by simulation of fluorescence due to release of Ca2+ from a spherical source, in a homogeneous three-dimensional space that contained components representing cytoplasmic molecules and Ca2+ removal processes. Total release current, as well as source diameter and noise level, was varied in the simulations. Derived release flux or current, calculated by volume integration of the derived flux density, estimated quite closely the current used in the simulation, while full width at half magnitude of the derived release flux was a good monitor of source size only at diameters >0. 7 micrometers. On an average of 157 sparks of amplitude >2 U resting fluorescence, located automatically in a representative voltage clamp experiment, the algorithm reported a release current of 16.9 pA, coming from a source of 0.5 micrometer, with an open time of 6.3 ms. Fewer sparks were obtained in permeabilized fibers, so that the algorithm had to be applied to individual sparks or averages of few events, which degraded its performance in comparable tests. The average current reported for 19 large sparks obtained in permeabilized fibers was 14.4 pA. A minimum estimate, derived from the rate of change of dye-bound Ca2+ concentration, was 8 pA. Such a current would require simultaneous opening of between 8 and 60 release channels with unitary Ca2+ currents of the level recorded in bilayer experiments. Real sparks differ from simulated ones mainly in having greater width. Correspondingly, the algorithm reported greater spatial extent of the source for real sparks. This may again indicate a multichannel origin of sparks, or could reflect limitations in spatial resolution.

Different effects of two gold compounds on muscle contraction, membrane potential and ryanodine receptor

Effects of gold sodium thiomalate and NaAuCl 4 on skeletal muscle function were studied using intact single fibres of frog skeletal muscle and fragmented sarcoplasmic reticulum prepared from frog and rabbit skeletal muscles. Gold sodium thiomalate at a concentration of 500 μM decreased tension amplitude by 27% and resting membrane potential by 5.3% after 30 and 22 min, respectively. The duration of tetanus tension was markedly shortened by 500 μM gold sodium thiomalate. When 10 μM NaAuCl 4 was applied to gold sodium thiomalate-pretreated fibres, the fibres lost the ability to contract upon electrical stimulation, similar to the effects of 10 μM NaAuCl 4 alone. In the presence of thiomalic acid, on the other hand, NaAuCl 4 did not completely block tetanus tension even at 50 μM. Thiomalic acid also inhibited NaAuCl 4-induced membrane depolarization. These findings suggest that thiomalate masks the effects of gold ion on muscle function. When sarcoplasmic reticulum vesicles were incorporated into lipid bilayers, exposure of the cis side of the Ca 2+-release channel to 100 μM gold sodium thiomalate rapidly increased the open probability of the channel 3.3-fold, from 0.032 in controls to 0.105, with an increase in number of open events and a decrease in mean closed time. The ability of NaAuCl 4 to activate the Ca 2+-release channel was much stronger than that of gold sodium thiomalate. Only 1 μM NaAuCl 4 was enough to activate the channel and this gold was effective from either side of the channel. These results suggest that gold sodium thiomalate could be used as an antirheumatic drug without considering severe side-effects on skeletal muscle. Coexistent thiomalate probably contributes to protection of muscle function from side-effects of gold ion.

Backward Movements of Cross-Bridges by Application of Stretch and by Binding of MgADP to Skeletal Muscle Fibers in the Rigor State as Studied by X-Ray Diffraction

The effects of the applied stretch and MgADP binding on the structure of the actomyosin cross-bridges in rabbit and/or frog skeletal muscle fibers in the rigor state have been investigated with improved resolution by x-ray diffraction using synchrotron radiation. The results showed a remarkable structural similarity between cross-bridge states induced by stretch and MgADP binding. The intensities of the 14.4- and 7.2-nm meridional reflections increased by ∼23 and 47%, respectively, when 1 mM MgADP was added to the rigor rabbit muscle fibers in the presence of ATP-depletion backup system and an inhibitor for muscle adenylate kinase or by ∼33 and 17%, respectively, when rigor frog muscle was stretched by ∼4.5% of the initial muscle length. In addition, both MgADP binding and stretch induced a small but genuine intensity decrease in the region close to the meridian of the 5.9-nm layer line while retaining the intensity profile of its outer portion. No appreciable influence was observed in the intensities of the higher order meridional reflections of the 14.4-nm repeat and the other actin-based reflections as well as the equatorial reflections, indicating a lack of detachment of cross-bridges in both cases. The changes in the axial spacings of the actin-based and the 14.4-nm-based reflections were observed and associated with the tension change. These results indicate that stretch and ADP binding mediate similar structural changes, being in the correct direction to those expected for that the conformational changes are induced in the outer portion distant from the catalytic domain of attached cross-bridges. Modeling of conformational changes of the attached myosin head suggested a small but significant movement (about 10–20°) in the light chain-binding domain of the head toward the M-line of the sarcomere. Both chemical (ADP binding) and mechanical (stretch) intervensions can reverse the contractile cycle by causing a backward movement of this domain of attached myosin heads in the rigor state.

A repetitive mode of activation of discrete Ca2+ release events (Ca2+ sparks) in frog skeletal muscle fibres.

1. Ca2+ release events (Ca2+ 'sparks'), which are believed to arise from the opening of a sarcoplasmic reticulum (SR) Ca2+ release channel or a small cluster of such channels that act as a release unit, have been measured in single, frog (Rana pipiens) skeletal muscle fibres. 2. Under conditions of extremely low rates of occurrence of Ca2+ sparks we observed, within individual identified triads, repetitive Ca2+ release events which occurred at a frequency more than 100-fold greater than the prevailing average event rate. Repetitive sparks were recorded during voltage-clamp test depolarizations after a brief (0.3-2 s) repriming interval in fibres held at 0 mV and in chronically depolarized, 'notched' fibres. 3. These repetitive events are likely to arise from the re-opening of the same SR Ca2+ release channel or release unit operating in a repetitive gating mode ('rep-mode'), rather than from the random activation of multiple, independent channels or release units within a triad. A train of rep-mode events thus represents a series of Ca2+ sparks arising from a single location within the fibre. Rep-mode events are activated among different triads in a random manner after brief repriming. The frequency of repetitive events among all identified events during voltage-clamp depolarization to 0 mV after brief repriming was 3.9 +/- 1.3 %. The occurrence of repetitive events was not related to exposure of the fibre to laser illumination. 4. The events observed within a rep-mode train exhibited a relatively uniform amplitude. Analysis of intervals between identified events in triads exhibiting rep-mode trains indicated similar variations of fluorescence as in neighbouring, quiescent triads, suggesting there was not a significant number of small, unidentified events at the triads exhibiting rep-mode activity. 5. The distribution of rep-mode interspark intervals exhibited a paucity of events at short intervals, consistent with the need for recovery from inactivation before activation of the next event in a repetitive train. The mean interspark interval of repetitive sparks during voltage-clamp depolarizations was 88 +/- 5 ms, and was independent of membrane potential. 6. The individual Ca2+ sparks within a rep-mode train were similar in average amplitude and spatiotemporal extent to singly occurring sparks, suggesting a common mechanism for termination of the channel opening(s) underlying both types of events. The average properties of the sparks did not vary during a train. The relative amplitude of a spark within a rep-mode was not correlated with its rise time. 7. Repetitive Ca2+ release events represent a mode of gating of SR Ca2+ release channels which may be significant during long depolarizations and which may be influenced by the biochemical state of the SR ryanodine receptor Ca2+ release channels.

Time course of individual Ca2+ sparks in frog skeletal muscle recorded at high time resolution.

Discrete Ca2+ release events (Ca2+ "sparks") were recorded in cut segments of single frog skeletal muscle fibers using a video-rate laser-scanning confocal microscope operating in line-scan mode (63 microseconds per line). Fibers loaded with the Ca2+ indicator fluo-3 were voltage clamped at a holding potential of 0 mV, briefly reprimed at -90 mV, and then strongly depolarized with a large test pulse to activate any reprimed voltage sensors. Using this high time resolution system, it was possible to record individual Ca2+ sparks at approximately 30-fold higher time resolution than previously attained. The resulting new experimental data provides a means of characterizing the time course of fluorescence during the brief (a few milliseconds) rising phase of a spark, which was not possible with the previously used 1.5-2 ms per line confocal systems. Analysis of the time course of individual identified events indicates that fluorescence begins to rise rather abruptly at the start of the spark, continues to rise at a slightly decreasing rate to a relatively sharp peak, and then declines along a quasi-exponential time course. The mean rise time of 198 sparks was 4.7 +/- 0.1 ms, and there was no correlation between rise time and peak amplitude. Average sparks constructed by temporally and spatially superimposing and summing groups of individual sparks having similar rise times gave a lower noise representation of the sparks, consistent with the time course of individual events. In theory, the rising phase of a spark provides a lower bound estimation of the time that Ca2+ ions are being released by the sarcoplasmic reticulum Ca2+ channel(s) generating the spark. The observed time course of fluorescence suggests that the Ca2+ release underlying a spark could continue at a fairly constant rate throughout the rising phase of the spark, and then stop rather abruptly at the time of the peak.

3PB013 T処理したカエル骨格筋スキンドファイバーのX線回折

3PB013 T処理したカエル骨格筋スキンドファイバーのX線回折

Effects of ramp shortening during linear phase of relaxation on [Ca2+]i in intact skeletal muscle fibers.

The effects of shortening distance at Vu, the unloaded shortening speed, and filament overlap on the amount of extra Ca2+ released during relaxation in muscle, as indicated by the bump area, were studied. Single, intact frog skeletal muscle fibers at 3 degreesC were used. The myoplasmic free Ca2+ concentration ([Ca2+]i) was estimated by using fura 2 salt injected into the myoplasm. Ramps were applied, either at full overlap with different sizes or at varying overlaps with a fixed size, in the linear phase of relaxation. At full overlap, a plot of bump area vs. ramp size was fit by using a sigmoidal curve with one-half of the bump area equal to 25.9 nm. With a fixed ramp size of 100 nm/half-sarcomere, the plot of bump area vs. mean sarcomere length (SLm) was fit by a straight line intersecting the SLm axis at approximately 3.5 micrometers, close to just no overlap. The results suggest that the transition in the distribution of attached cross bridges from the isometric case to one appropriate for unloaded shortening at Vu is completed within 50 nm/half-sarcomere and support the view that attached cross bridges in the overlap zone influence the affinity of Ca2+ for troponin C in the thin filament.

Effects of caffeine and adenine nucleotides on Ca2+ release by the sarcoplasmic reticulum in saponin-permeabilized frog skeletal muscle fibres.

1. The effect of caffeine and adenine nucleotides on the sarcoplasmic reticulum (SR) Ca2+ release mechanism was investigated in permeabilized frog skeletal muscle fibres. Caffeine was rapidly applied and the resulting release of Ca2+ from the SR detected using fura-2 fluorescence. Decreasing the [ATP] from 5 to 0.1 mM reduced the caffeine-induced Ca2+ transient by 89 +/- 1.4% (mean +/- s.e.m., n = 16), while SR Ca2+ uptake was unaffected. 2. The dependence of caffeine-induced Ca2+ release on cytosolic [ATP] was used to study the relative ability of other structurally related compounds to substitute for, or compete with, ATP at the adenine nucleotide binding site. It was found that AMP, ADP and the non-hydrolysable analogue adenylyl imidodiphosphate (AMP-PNP) partially substituted for ATP, although none was as potent in facilitating the Ca2+-releasing action of caffeine. 3. Adenosine reversibly inhibited caffeine-induced Ca2+ release, without affecting SR Ca2+ uptake. Five millimolar adenosine markedly reduced the amplitude of the caffeine-induced Ca2+ transient by 64 +/- 4% (mean +/- s.e.m., n = 11). The degree of inhibition was dependent upon the cytosolic [ATP], suggesting that adenosine may act as a competitive antagonist at the adenine nucleotide binding site. 4. These data show that (i) the sensitivity of the in situ SR Ca2+ channel to caffeine activation is strongly dependent upon the cytosolic [ATP], (ii) the number of phosphates attached to the 5' carbon of the ribose ring influences the efficacy of the ligand, and (iii) removal of a single phosphate group transforms AMP from a partial agonist, to adenosine, which acts as a competitive antagonist under these conditions.

Supercharging Accelerates T-Tubule Membrane Potential Changes in Voltage Clamped Frog Skeletal Muscle Fibers

In voltage-clamp studies of single frog skeletal muscle fibers stained with the potentiometric indicator 1-(3-sulfonatopropyl)-4-[ β[2-(di- n-octylamino)-6-naphthyl]vinyl]pyridinium betaine (di-8 ANEPPS), fluorescence transients were recorded in response to both supercharging and step command pulses. Several illumination paradigms were utilized to study global and localized regions of the transverse tubule system (T-system). The rising phases of transients obtained from global illumination regions showed distinct accelerations when supercharging pulses were applied (95% of steady-state fluorescence achieved in 1.5 ms with supercharging pulses versus 14.6 ms with step pulses). When local transients were recorded at the edge of the muscle fiber, their kinetics resembled those of the applied waveform, but a similar relationship was not observed in transients from regions near the edge chosen to minimize the surface membrane contribution. We developed a model of the T-system capable of simulating membrane potential changes as a function of time and distance along the T-system cable and the associated fluorescence changes in regions corresponding to the experimental illumination strategies. A critical parameter was the access resistance term, for which values of 110–150 Ω·cm 2 were adequate to fit the data. The results suggest that the primary mechanism through which supercharging pulses boost the kinetics of T-system voltage changes most likely involves their compensating the voltage attenuation across the access resistance at the mouth of the T-tubule.

Fast voltage gating of Ca2+ release in frog skeletal muscle revealed by supercharging pulses.

1. In single frog skeletal muscle fibres, we utilized supercharging voltage clamp command pulses to boost the rate of depolarization in the transverse tubular system (T-system) such that 95 % of steady-state potential is achieved in < 2 ms (as indicated by fluorescent potentiometric dye signals detected from a global illumination region). Signals detected near the edge of muscle fibres indicate that peripheral regions of the T-system are not significantly overcompensated under these conditions. 2. We explored the impact of accelerating T-system depolarization on voltage-dependent events of excitation-contraction (E-C) coupling by measuring charge movement currents (CMCs) and Ca2+ fluorescence transients in response to both supercharging and conventional step pulses. 3. When compared with CMCs elicited by step pulses, supercharging CMCs are larger, and their kinetics more closely resemble those of gating current records reported for ionic channels. Furthermore, they decay bi-exponentially (tau fast range, 1.3-1.8 ms; tau slow range, 7.3-11.9 ms), whereas step CMCs fall with a single exponential time course (tau range, 12.5-26.7 ms). 4. Similarly, supercharging produces a distinct acceleration in Ca2+ release transients, which show little evidence of the voltage-dependent onset latencies previously encountered using step pulses. 5. The use of this novel methodology in skeletal muscle unveils a previously undetected component of charge movement, the rapid, voltage-dependent recruitment of which may provide the basis for understanding the fast gating of physiological E-C coupling.

Model of sarcomeric Ca2+ movements, including ATP Ca2+ binding and diffusion, during activation of frog skeletal muscle.

Cannell and Allen (1984. Biophys. J. 45:913-925) introduced the use of a multi-compartment model to estimate the time course of spread of calcium ions (Ca2+) within a half sarcomere of a frog skeletal muscle fiber activated by an action potential. Under the assumption that the sites of sarcoplasmic reticulum (SR) Ca2+ release are located radially around each myofibril at the Z line, their model calculated the spread of released Ca2+ both along and into the half sarcomere. During diffusion, Ca2+ was assumed to react with metal-binding sites on parvalbumin (a diffusible Ca2+- and Mg2+-binding protein) as well as with fixed sites on troponin. We have developed a similar model, but with several modifications that reflect current knowledge of the myoplasmic environment and SR Ca2+ release. We use a myoplasmic diffusion constant for free Ca2+ that is twofold smaller and an SR Ca2+ release function in response to an action potential that is threefold briefer than used previously. Additionally, our model includes the effects of Ca2+ and Mg2+ binding by adenosine 5'-triphosphate (ATP) and the diffusion of Ca2+-bound ATP (CaATP). Under the assumption that the total myoplasmic concentration of ATP is 8 mM and that the amplitude of SR Ca2+ release is sufficient to drive the peak change in free [Ca2+] (Delta[Ca2+]) to 18 microM (the approximate spatially averaged value that is observed experimentally), our model calculates that (a) the spatially averaged peak increase in [CaATP] is 64 microM; (b) the peak saturation of troponin with Ca2+ is high along the entire thin filament; and (c) the half-width of Delta[Ca2+] is consistent with that observed experimentally. Without ATP, the calculated half-width of spatially averaged Delta[Ca2+] is abnormally brief, and troponin saturation away from the release sites is markedly reduced. We conclude that Ca2+ binding by ATP and diffusion of CaATP make important contributions to the determination of the amplitude and the time course of Delta[Ca2+].

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.

Evidence for Two Distinct Cross-Bridge Populations in Tetanized Frog Muscle Fibers Stretched with Moderate Velocities

When tetanized frog skeletal muscle fibers are subjected to moderate-velocity stretches (<1L0/s), the tension developed above the level of isometric tension starts to decay after a sudden reduction of stretch velocity by more than 40-50%, though the fibers are still being stretched. We analysed the decay of tension response caused by the sudden reduction of stretch velocity, by applying three different types of stretch to a tetanized fiber, i.e., a 1.5% stretch with velocity V1(stretch-1), a 1.5% stretch with velocity V2< V1(stretch-2), and a 3% stretch consisting of stretch-1 and stretch-2 applied in succession (stretch-3) and comparing the corresponding tension responses, TR-1, TR-2, and TR-3. It was found that TR-3 to stretch-3 resulted from algebraical summation of TR-1 to the preceding stretch-1 and TR-2 to the subsequent stretch-2. These results can be accounted for by assuming two distinct cross-bridge populations in stretched fibers.