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The possibility that weakly binding bridges are attached to actin in the absence of Ca2+ under physiological conditions was investigated by studying the force response of unstimulated intact muscle fibres of the frog to fast ramp stretches. The force response during the stretching period is divided into two phases: phase 1, coincident with the acceleration period of the sarcomere length change and phase 2, synchronous with sarcomere elongation at constant speed. The phase 1 amplitude increases linearly with the stretching speed in all the range tested, while phase 2 increases with the speed but reaches a plateau level at about 50 x 10(3) nm/half sarcomere per second. The analysis of data shows that phase 1, which corresponds to the initial 5-10 nm/half sarcomere of elongation, is very likely a pure viscous response; its amplitude increases with sarcomere length and it is not affected by the electrical stimulation of the fibre. Phase 2 is a viscoelastic response with a relaxation time of the order of 1 ms; its amplitude increases with sarcomere lengths and with the stimulation. These data suggest that weakly binding bridges are not present in a significant amount in unstimulated intact fibres.

Equatorial X-ray diffraction techniques have been successfully applied to the intact single muscle fibre preparation under length clamp and "fixed end" conditions. 10 and 11 intensity changes and stiffness have been measured in the same preparation. Under isometric conditions, equatorial signals and stiffness led force by 14-20ms during the rise of tetanic tension. During relaxation, stiffness and equatorial signals lagged force. The time course of the intensity changes suggests a low force crossbridge state is present to a greater extent during the rise of tetanic tension and during relaxation than at the tetanus plateau. During isotonic shortening at Vmax, stiffness fell to 30% of its isometric level, while equatorial signals fell to 60%. Since stiffness and equatorial signals are thought to detect attached crossbridges, either the average stiffness per attached bridge measured at 4kHz during shortening is less than at the plateau, or the relation between equatorial intensities and the proportion of attached crossbridges during isotonic shortening differs from that measured under isometric conditions. Active tension also affects the lattice spacing. The myosin lattice was compressed during the development of longitudinal force. This implies a radial component of crossbridge tension. The lattice compression was smaller in a compressed lattice and larger in an expanded lattice.

Several experimental results (Schoenberg, M. 1988. Biophys. J. 54:135-148) have shown that the force response of relaxed skinned muscle fibers to fast stretches arises from the presence of cross-bridges rapidly cycling between attached and detached states. These bridges were identified with the M.ATP<-->AM.ATP and M.ADP.Pi<-->AM.ADP.Pi states seen in solution and are commonly referred to as weakly binding bridges. In this paper we have investigated the possibility that weakly binding bridges are also present in resting intact muscle fibers. The force response to fast stretches can be accounted for by assuming the presence in the fiber of a viscous and a viscoelastic passive component arranged in parallel. None of these components has the properties previously attributed to weakly binding bridges. This shows that in intact resting fibers there is no mechanical evidence of attached cross-bridges, suggesting that, under physiological conditions, either the M.ATP or M.ADP.Pi states have a negligibly small affinity for actin or the AM.ATP and AM.ADP.Pi cross-bridge states are unable to bear tension and contribute to fiber stiffness.

Time-resolved lattice spacing changes were measured (10-millisecond time resolution) by x-ray diffraction of synchrotron radiation in single intact muscle fibers of the frog Rana temporaria undergoing electrically stimulated tension development during application of stretches and releases. Ramp releases, which decreased fiber length at constant speed, caused a lattice expansion. After the ramp, increasing tension during recovery was accompanied by lattice compression. Ramp stretches caused a compression of the lattice. While the fiber was held at a constant length after the stretch, tension decreased and lattice spacing increased. These observations demonstrate the existence of a previously undetected radial component of the force generated by a cycling crossbridge. At sarcomere lengths of 2.05 to 2.2 micrometers, the radial force compresses the myofilament lattice. Hence, the myofilament lattice does not maintain a constant volume during changes in force.

1. The relation between sarcomere length and steady tetanic tension was determined at 10-12 degrees C for 70-80 microns long length-clamped segments of single fibres isolated from the tibialis anterior muscle of the frog, in normal and hypertonic or hypotonic Ringer solutions. 2. The tension depression and potentiation observed in hypertonic and hypotonic Ringers solutions varied with sarcomere length, so that, as opposed to myofilament overlap predictions, the optimum length for tension development was shorter in hypertonic Ringer solution and longer in hypotonic Ringer solution than in normal Ringer solution. As the fibres were stretched from 1.96 to 2.24 microns sarcomere length, both tension depression in hypertonic Ringer solution and tension potentiation in hypotonic Ringer solution increased by 9 and 5%, respectively. 3. Within this range of sarcomere lengths the length-stiffness relation in hypotonic and in hypertonic Ringer solutions exhibit little or no change relative to that in normal Ringer solution. 4. The results indicate that separation between the thick and the thin myofilaments influences the mechanism of force generation. There is an optimum interfilament distance (10-12 nm surface to surface between the thick and the thin filaments) for tension production. In isotonic Ringer solution, this corresponds to the interfilament distance at sarcomere lengths around 2.10 microns. The force per attached cross-bridge, rather than their number, appears to decrease as the interfilament distance is brought above or below the optimum length. Even if this effect is moderate in isotonic Ringer solution, it should be taken into account in models of the force-generation mechanism.

The time course of the contractile process was investigated in the presence of AR-L 115BS, a twitch potentiator which is thought to increase the rate of the Ca2+ binding by troponin and to improve the Ca2+ mobilization from the sarcoplasmic reticulum. AR-L 115BS increased markedly the rate of development of the force-velocity (T-V) relation and of the isometric tetanic tension. The effect on the rate of development of the T-V relation was however substantially more intense than that on the speed of rise of tetanic tension, thus reducing considerably the isometric tension level at which the T-V relation attained its final characteristics. The velocity of shortening under zero load and the degree of curvature of the T-V relation were not affected by AR-L 115BS. These findings support the view that the development of the T-V relation and the rise of the isometric tetanic tension measure the time course of two different processes.

The relation between sarcomere length, tension and time course of tension development in twitch and tetanic contractions at 20 degrees C was determined for isolated fibres from the semitendinosus muscle of the frog (Rana esculenta). In twenty fibres at about 2.15 micron sarcomere length, the peak twitch tension, the maximum tetanic tension and the twitch/tetanus ratio ranged, respectively, from 0.22 to 1.6 kg/cm2, from 2.13 o 3.96 kg/cm2 an from 0.07 to 0.53. The peak twitch tension was found to be: i) directly correlated with the twitch/tetanus ratio and the time to the peak of the first derivative of the twitch tension, ii) inversely correlated with the time to the peak of the first derivative of tetanic tension. No significant correlation was found between the maximal tetanic tension and the peak twitch tension or the twitch/tetanus ratio. Peak twitch tension and twitch/tetanus ratio were not correlated with the fibre cross-sectional area which ranged from 1.052 to 6,283 micron2. Sarcomere length-tension curves for twitch and tetanic isometric contractions at 20 degrees C were determined in twelve fibres. Increases in sarcomere length from about 2.15 to 2.85 micron produced, depending on the peak twitch tension or the twitch/tetanus ratio at about 2.15 micron, either decrease and no change or increase in peak twitch tension, but constantly enhanced the twitch/tetanus ratio and the degree of this potentiation was inversely correlated with the twitch/tetanus ratio at 2.15 micron. Increase in sarcomere length above 2.15 micron did not alter the course of the early development of twitch and tetanic tensions, reduced considerably the variation in peak twitch tension and twitch/tetanus ratio, without altering that of tetanic tension and swamped the correlation between the peak twitch tension and the time to peak of the differentiated twitch tension. However, the peak twitch tension at about 2.85 micron resulted to be directly correlated with the peak twitch tension at about 2.15 micron and in addition the relative length-dependent change in the time of the peak of the first derivative of the twitch tension resulted to be directly correlated with the relative length-dependent change in the peak twitch tension. It is concluded that both the duration of the active state and the rate factors of activation contribute to the determining of the large variation in peak twitch tension at about 2.15 micron, whereas the length-dependent increase in twitch/tetanus ratio appears to be mainly determined by prolongation of the active state duration.

A circuit specially designed for use with fast capacitance-gauge force transducer suitable for experiments in single skeletal muscle fibre, is presented. The circuit is a modified solid-state version (with improved characteristics) of the Haines (3) circuit. The electronics is simple and small enough to be placed very close to the transducer.

1. The force-velocity (P-V) relation for normal or NO-3 treated single fibres isolated from the semitendinosus muscle of the frog was determined at given times during the rise of tension and the plateau of isometric tetani. Experiments were made at about 2.25 micron sarcomere length and at constant temperatures, from 3 to 4.5 degrees C and from 19 to 21 degrees C. The controlled-velocity release method was used. 2. During the rise of tension, at any initial tension higher than about 0.2 P0, the lowest release velocity required to drop the tension to zero was the same as at the tetanic plateau, independent of the temperature and the presence of NO-3 ions in the bathing solution. 3. The degree of activation (measured by the steady force exerted at a given velocity of shortening lower than V0) increased with time, but attained its steady-state level before isometric tension. 4. At about 20 degrees C, frog muscle fibres at about 2.2 micron sarcomere length were only partially activated after a single stimulus. 5. NO-3 ions did not affect the steady-state P-V relation. At about 20 degrees C, NO-3 ions increased the rate of development of activation. Potentiation of the twitch contraction was due at least in part to this mechanism. 6. The 'relative' P-V relation appears to be independent of both the time after start of stimulation and the presence of NO-3 ions in the bathing solution. 7. The results are discussed in terms of the sliding filament model of Huxley (1957), assuming that either the number of actin sites available for cross-bridge formation, or the value of the rate constant for making of cross-bridges, is time dependent.

1. The force-velocity (P-V) relation from a single fibres isolated from the semitendinosus muscle of the frog was determined at pre-set times during the rise of tension and the plateau of isometric tetani. The controlled-velocity release method was used. Experiments were performed at about 2.25 micrometers sarcomere length and at 3-4 degrees C or at 19-21 degrees C. 2. Replacing H2O with D2O resulted in a rapid large reduction of the peak twitch tension and of the speed of development of twitch and tetanic tensions. The tetanic tension (P0) was usually reduced, in certain fibres to as low as 5% of the value in H2O-Ringer solution. 3. The depression of twitch and tetanus characteristics was followed by a recovery, the duration of which varied greatly in different fibres. During the recovery period previous conditioning activity potentiated the tetanus characteristics. 4. After the end of the recovery period in D2O-Ringer solution both the peak twitch tension and the speed of development of tetanic tension was still greatly depressed, whereas the value of P0 was slightly greater than in H2O-Ringer. The speed of rise of isometric tension after a quick release imposed at the tetanus plateau was reduced in D2O-Ringer, usually to about 50% of the value in H2O-Ringer. 5. D2O increased the development time of the P-V relation and produced a conspicuous increase in the degree of its curvature. The value of V0 (the velocity of shortening at zero load) was not significantly depressed by D2O and it was the same independent both of the time after the beginning of stimulation and of the isometric tension at which the measurement was made. The P-V relation attained its final characteristics before the isometric tension reached the plateau. During the recovery period in D2O-Ringer, at the plateau of isometric tetani of different size, the relative force exerted at a given velocity of shortening was constant. 6. In D2O-treated fibres, NO3- and caffeine (i) potentiated the peak twitch tension and the speed of development of tetanic tension without affecting significantly the speed of the redevelopment of tension after a quick release imposed at the tetanus plateau and (ii) reduced the development time of the P-V relation, but did not affect either the degree of its curvature or the value of V0 and P0. 7. The results are discussed by assuming that the release of Ca2+ from the sarcoplasmic reticulum is a rate-limiting process for the development of activation and in turn for the development of isometric tension. In terms of the cross-bridge model of Huxley (1957), the time or Ca2+-dependent factor of activation appears to be the recruitment of actin sites for cross-bridge formation, whereas the value of the rate constants regulating the cross-bridge kinetics appears to be time and Ca2+-independent.

Whereas the mechanical behavior of fully activated fibers can be explained by assuming that attached force-producing crossbridges exist in at least two configurations, one exerting more force than the other (Huxley A. F., and R. M. Simmons. 1971. Nature [Lond.]. 233:533-538), and the behavior of relaxed fibers can be explained by assuming a single population of weakly binding rapid-equilibrium crossbridges (Schoenberg, M. 1988. Biophys. J. 54:135-148), it has not been possible to explain the transition between rest and activation in these terms. The difficulty in explaining why, after electrical stimulation of resting intact frog skeletal muscle fibers at 1-5 degrees C, force development lags stiffness development by more than 15 ms has led a number of investigators to postulate additional crossbridge states. However, postulation of an additional crossbridge state will not explain the following three observations: (a) Although the lag between force and stiffness is very different after stimulation, during the redevelopment of force after an extended period of high velocity shortening, and during relaxation of a tetanus, nonetheless, the plots of force versus stiffness in each of these cases are approximately the same. (b) When the lag between stiffness and force during the rising phase of a twitch is changed nearly fourfold by changing temperature, again the plot of force versus stiffness remains essentially unchanged. (c) When a muscle fiber is subjected to a small quick length change, the rate constant for the isometric force recovery is faster when the length change is applied during the rising phase of a tenanus than when it is applied on the plateau. We have been able to explain all the above findings using a model for force production that is similar to the 1971 model of Huxley and Simmons, but which makes the additional assumption that the force-producing transition envisioned by them is a cooperative one, with the back rate constant of the force-producing transition decreasing as more crossbridges attach.

Single fibers, isolated intact from frog skeletal muscles, were held firmly very near to each end by stiff metal clasps fastened to the tendons. The fibers were then placed horizontally between two steel hooks inserted in eyelets of the tendon clasps. One hook was attached to a capacitance gauge force transducer (resonance frequency up to approximately 50 kHz) and the other was attached to a moving-coil length changer. This allowed us to impose small, rapid releases (complete in less than 0.15 ms) and high frequency oscillations (up to 13 kHz) to one end of a resting or contracting fiber and measure the consequences at the other end with fast time resolution at 4 to 6 degrees C. The stiffness of short fibers (1.8-2.6 mm) was determined directly from the ratio of force to length variations produced by the length changer. The resonance frequency of short fibers was so high (approximately 40 kHz) that intrinsic oscillations were not detectably excited. The stiffness of long fibers, on the other hand, was calculated from measurement of the mechanical resonance frequency of a fiber. Using both short and long fibers, we measured the sinusoids of force at one end of a contracting fiber that were produced by relatively small sinusoidal length changes at the other end. The amplitudes of the sinusoidal length changes were small compared with the size of step changes that produce nonlinear force-extension relations. The sinusoids of force from long fibers changed amplitude and shifted phase with changes in oscillation frequency in a manner expected of a transmission line composed of mass, compliance, and viscosity, similar to that modelled by (Ford, L. E., A. F. Huxley, and R. M. Simmons, 1981, J. Physiol. (Lond.), 311:219-249). A rapid release during the plateau of tetanic tension in short fibers caused a fall in force and stiffness, a relative change in stiffness that putatively was much smaller than that of force. Our results are, for the most part, consistent with the cross-bridge model of force generation proposed by Huxley, A. F., and R. M. Simmons (1971, Nature (Lond.), 213:533-538). However, stiffness in short fibers developed markedly faster than force during the tetanus rise. Thus our findings show the presence of one or more noteworthy cross-bridge states at the onset and during the rise of active tension towards a plateau in that attachment apparently is followed by a relatively long delay before force generation occurs. A set of equations is given in the Appendix that describes the frequency dependence of the applied sinusoid and its response. This model predicts that frequency dependent changes can be used as a measure of a change in stiffness.

Calcium transients, monitored by aequorin, and force were recorded simultaneously during tetanic contractions of isolated frog skeletal muscle fibers. Quick length changes were applied to the fibers during contractions at sarcomere lengths on the descending limb of the length-tension relationship. Previous experiments showed that regulatory Ca2+ binding sites are apparently saturated during a plateau of tetanic force development at these sarcomere lengths. However, quick releases of greater than 4 to 5% of fiber length produced a momentary fall in the calcium transient that followed a time course similar to the redevelopment of force. The fall in the Ca2+ transient after a release was maximum at striation spacings about half way along the descending limb (2.6-2.7 microns), which suggests it is not related to an increase in the number of Ca2+ binding sites distributed uniformly along the filaments. The effect was absent or barely detectable when highly stretched fibers were released during contraction. The fall in the Ca2+ transient was unrelated to the time during a tetanus that a release was made or to the velocity of the release. One explanation of these results is that complexes between actin and myosin are broken by a sudden reduction of length, and as they reform during the recovery of force the affinity of troponin for Ca2+ increases. Quick stretch had no effect on the rapid decay of Ca2+ transients, but stretch increased peak force and slowed relaxation for almost a second after the end of stimulation. Evidently the decrease in the rate of relaxation produced by stretch is unrelated to changes in the amount of Ca2+ released or the rate of Ca2+ removal, which supports suggestions that the kinetics of muscle relaxation are determined by more than one mechanism. The apparent increase in the overall duration of mechanical activity after stretch probably results from the longitudinal inhomogeneity in the duration of activity - known to occur during relaxation - coupled with the decreased compliance of stretched fibers.

The relation between stiffness and tension and the characteristics of the early tension recovery in response to applied small length step were studied both during tetanus rise and redevelopment of tension following a period of shortening at Vmax. Experiments were performed on single fibres isolated from tibialis anterior and lumbricalis muscles of the frog. Development of stiffness preceded that of tension during tension redevelopment, but the leading of stiffness was reduced to about one half of that found during the tetanus rise. The relation between relative stiffness and relative tension was the same either during tetanus rise and tension redevelopment. The speed of the early tension recovery in response to a step length change applied during the tension redevelopment was unchanged with respect to that found at the same tension during the tetanus rise. These results suggest that a cross-bridge state generating no force (or low force) may be a normal intermediate of the cross-bridge cycle even when the fibre is fully activated.

Stiffness measurements were performed during the rise, the plateau and the relaxation of tetanic contractions both in whole single muscle fibres and in tendon-free fibre segments under either fixed-end or length-clamp conditions. Fibres were isolated from the tibialis anterior muscle of the frog. Experiments were performed at 2-6 degrees C. Changes in length of tendon-free fibre segments were monitored by means of a "striation follower", an opto-electronic device which, during contraction, measured sarcomere displacement at the level of two selected regions of a fibre. Fast length perturbations imposed at one tendon end of a fibre during the plateau of tetanic contractions distribute uniformly along its length. During the tetanus rise stiffness led isometric tension in whole fibres under fixed conditions as well as in tendon-free fibre segments under length-clamp conditions. It was confirmed that a significant part of the unlinearity of T1 relations is determined by tendon compliance. During the isometric phase of relaxation in fixed-end tetani, the decline of tension led that of stiffness both in whole fibres and in tendon-free fibre segments. It is concluded that the shift observed between stiffness and tension during tetanus rise and relaxation represents a true specific event in the contractile process.

1. The relation between sarcomere length and tetanic tension was determined at 10-12 degrees C for 70-80 microns long segments of single fibres isolated from the tibialis anterior and semitendinosus muscles of the frog. Measurements of segment striation spacings were performed during fixed-end or length-clamp contractions by means of a laser light diffractometer. 2. At sarcomere lengths of around 2.10 microns tetanic tension rose promptly to a steady plateau, independent of the recording conditions. At greater sarcomere lengths under fixed-end conditions the tension rise occurred in two distinct stages: an initial rapid rise followed by a much slower creep. The tension creep was entirely abolished in length-clamp contractions. 3. The sarcomere length-tension diagram of length-clamped segments of tibialis anterior fibres exhibited a definite flat region between about 1.96 and 2.16 microns where tension varied by less than 1.5%. The highly linear descending limb reached zero tension at about 3.53 microns. The shift to the left by about 0.10 microns, with respect to the length-tension diagram of length-clamped segments of semitendinosus fibres, may be tentatively explained by assuming that thin filament lengths vary in different muscles. 4. The results are in agreement with those of a previous work by Gordon, Huxley & Julian (1966) and support the hypothesis (Huxley, 1957, 1980) that muscle tension is produced by simultaneous action of independent force generators, in proportion to the number of myosin bridges overlapped by actin filaments.

A principal step in the process leading to muscle contraction is the intracellular release of Ca2+. We have detected and compared some physical and chemical events that reflect Ca2+ release in contracting frog skeletal muscle cells, described the effects of some agents that are believed to alter intracellular Ca2+ release during contraction, and speculated about the role of Ca2+ release in influencing some of the mechanical properties of frog muscle. The specific physical features recorded were changes in striation spacing, myofibrillar orientation, and force development. The chemical feature was the relative change in intracellular [Ca2+] recorded as light emission from cells microinjected with the Ca2+-sensitive protein aequorin. The presence or absence of a correlation among these variables has been used (i) to evaluate the action of some agents thought to change intracellular Ca2+ release in excitation--contraction (E--C) coupling, (ii) to further substantiate the effects of cell length on Ca2+ release, and (iii) to examine some details of models for E--C coupling. The results showed that potentiating agents enhance and prolong intracellular Ca2+ release without changing the rate of Ca2+ removal during E--C coupling. This extra Ca2+ does not produce the same effect on contractions at all lengths. Contractility is inversely related to cell length, and Ca2+-induced activation is normally less than maximum not only at short lengths but also at optimal striation spacings.

M3 reflection intensity ([I.sub.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.sub.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.sub.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.sub.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.sub.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. skeletal muscle; X-ray diffraction; muscle mechanics; molecular motors; subfragment 1 structure

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 Ca2+ concentration. In the experiments reported here, we investigated the possible role of Ca2+ in static stiffness by comparing static stiffness measurements in the presence of Ca2+ release inhibitors (D600, Dantrolene, ²H2O) and cross-bridge formation inhibitors [2,3-butanedione monoxime (BDM), hypertonicity]. Both series of agents inhibited tension; however, only D600, Dantrolene, and ²H2O decreased at the same time static stiffness, whereas BDM and hypertonicity left static stiffness unaltered. These results indicate that Ca2+, 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. [ABSTRACT FROM AUTHOR]