Sarcomere length dispersion in single skeletal muscle fibers and fiber bundles.

Light diffraction patterns produced by single skeletal muscle fibers and small fiber bundles of Rana pipiens semitendinosus have been examined at rest and during tetanic contraction. The muscle diffraction patterns were recorded with a vidicon camera interfaced to a minicomputer. Digitized video output was analyzed on-line to determine mean sarcomere length, line intensity, and the distribution of sarcomere lengths. The occurrence of first-order line intensity and peak amplitude maxima at approximately 3.0 mum is interpreted in terms of simple scattering theory. Measurements made along the length of a singel fiber reveal small variations in calculated mean sarcomere length (SD about 1.2%) and its percent dispersion (2.1% +/- 0.8%). Dispersion in small multifiber preparations increases approximately linearly with fiber number (about 0.2% per fiber) to a maximum of 8-10% in large bundles. Dispersion measurements based upon diffraction line analysis are comparable to SDs calculated from length distribution histograms obtained by light micrography of the fiber. First-order line intensity decreases by about 40% during tetanus; larger multifibered bundles exhibit substantial increases in sarcomere dispersion during contraction, but single fibers show no appreciable dispersion change. These results suggest the occurrence of asynchronous static or dynamic axial disordering of thick filaments, with a persistence in long range order of sarcomere spacing during contraction in single fibers.     

Sarcomere length dependence of the force-velocity relation in single frog muscle fibers.

The force-velocity relation of single frog fibers was measured at sarcomere lengths of 2.15, 2.65, and 3.15 microns. Sarcomere length was obtained on-line with a system that measures the distance between two markers attached to the surface of the fiber, approximately 800 microns apart. Maximal shortening velocity, determined by extrapolating the Hill equation, was similar at the three sarcomere lengths: 6.5, 6.0, and 5.7 microns/s at sarcomere lengths of 2.15, 2.65, and 3.15 microns, respectively. For loads not close to zero the shortening velocity decreased with increasing sarcomere length. This was the case when force was expressed as a percentage of the maximal force at optimal fiber length or as a percentage of the sarcomere-isometric force at the respective sarcomere lengths. The force-velocity relation was discontinuous around zero velocity: load clamps above the level that kept sarcomeres isometric resulted in stretch that was much slower than when the load was decreased below isometric by a similar amount. We fitted the force-velocity relation for slow shortening (less than 600 nm/s) and for slow stretch (less than 200 nm/s) with linear regression lines. At a sarcomere length of 2.15 microns the slopes of these lines was 8.6 times higher for shortening than for stretch. At 2.65 and 3.15 microns the values were 21.8 and 14.1, respectively. At a sarcomere length of 2.15 microm, the velocity of stretch abruptly increased at loads that were 160-170% of the sarcomere isometric load, i.e., the muscle yielded. However, at a sarcomere length of 2.65 and 3.15 microm yield was absent at such loads. Even the highest loads tested (260%) resulted in only slow stretch.(ABSTRACT TRUNCATED AT 250 WORDS)     

Size changes in single muscle fibers during fixation and embedding.

During fixation of single muscles fibers with glutaraldehyde, the volume of the fiber shrinks 20%, recovers in rinse and osmium tetroxide to near normal volume and shrinks 20% again when staining with uranyl acetate. This suggest that osmotic properties of membranes may not have been completely lost during fixation, post-fixation and en bloc staining. Dehydration in ethanol and propylene oxide produces a further 10% shrinkage in volume. Infiltration and embedding with Epon causes an additional 15% change in volume. This gives a total shrinkage in volume of 45% which is nearly twice that of the apparent shrinkage in the volume of the myosin lattice as determined by electron microscopy.     

Stepwise length changes in single invertebrate thick filaments

Previous experiments on thick filaments of the anterior byssus retractor muscle of Mytilus and the telson-levator muscle of Limulus polyphemus have shown large, reversible length changes up to 23% and 66% of initial length, respectively, within the physiological tension range. Using nanofabricated cantilevers and newly developed high-resolution detection methods, we investigated the dynamics of isolated Mytilus anterior byssus retractor muscle thick filaments. Single thick filaments were suspended between the tips of two microbeams oriented perpendicular to the filament axis: a deflectable cantilever and a stationary beam. Axial stress was applied by translating the base of the deflectable nanolever away from the stationary beam, which bent the nanolever. Tips of flexible nanolevers and stationary beam were imaged onto a photodiode array to track their positions. Filament shortening and lengthening traces, obtained immediately after the motor had imposed stress on the filament, showed steps and pauses. Step sizes were 2.7 nm and integer multiples thereof. Steps of this same size paradigm have been seen both during contraction of single sarcomeres and during active interaction between single isolated actin and myosin filaments, raising the question whether all of these phenomena might be related.     

Lattice spacing changes accompanying isometric tension development in intact single muscle fibers.

The myosin lattice spacing of single intact muscle fibers of the frog, Rana temporaria, was studied in Ringer's solution (standard osmolarity 230 mOsm) and hyper- and hypotonic salines (1.4 and 0.8 times standard osmolarity respectively) in the relaxed state, during "fixed end" tetani, and during shortening, using synchrotron radiation. At standard tonicity, a tetanus was associated with an initial brief lattice expansion (and a small amount of sarcomere shortening), followed by a slow compression (unaccompanied by sarcomere length changes). In hypertonic saline (myosin lattice compressed by 8.1%), these spacing changes were suppressed, in hypotonic saline (lattice spacing increased by 7.5%), they were enhanced. During unloaded shortening of activated fibers, a rapid lattice expansion occurred at all tonicities, but became larger as tonicity was reduced. This expansion was caused in part by the change in length of the preparation, but also by a recoil of a stressed radial compliance associated with axial force. The lattice spacing during unloaded shortening was equal to or occasionally greater than predicted for a relaxed fiber at that sarcomere length, indicating that the lattice compression associated with activation is rapidly reversed upon loss of axial force. Lattice recompression occurred upon termination of shortening under standard and hypotonic conditions, but was almost absent under hypertonic conditions. These observations indicate that axial cross-bridge tension is associated with a compressive radial force in intact muscle fibers at full overlap; however, this radial force exhibits a much greater sensitivity to lattice spacing than does the axial force.     

Fura-2 diffusion and its use as an indicator of transient free calcium changes in single striated muscle cells

Two questions bearing on the use of fura-2 to measure transient changes in intracellular Ca2+ concentration have been addressed. To investigate fura-2 intracellular binding, the amounts of fura-2 and [14C]glycine in Balanus nubilus myofibrillar bundles after loading were determined and their intracellular apparent diffusion constants measured. No significant fura-2 immobilisation occurs under the conditions used. The apparent diffusion constant for fura-2 in aqueous solution was determined. The relationship between half-time for relaxation of force and fura-2 fluorescence transients, and intracellular fura-2 concentration, in voltage-clamped single muscle fibres was examined. Significant buffering of the Ca2+ transient occurred at fura-2 concentrations above approximately 6 microM.     

Muscle fiber and tendon length changes in the human vastus lateralis during slow pedaling.

Muscle fascicle lengths of vastus lateralis (VL) muscle were measured in five healthy men during slow pedaling to investigate the interaction between muscle fibers and tendon. Subjects cycled at a pedaling rate of 40 rpm (98 W). During exercise, fascicle lengths changed from 91 +/- 7 (SE) to 127 +/- 5 mm. It was suggested that fascicles were on the descending limb of their force-length relationship. The average shortening velocity of fascicle was greater than that of muscle-tendon complex in the first half of the knee extension phase and was less in the second half. The maximum shortening velocity of fascicle in the knee extension phase was less than that of muscle-tendon complex by 22 +/- 9%. These discrepancies in velocities were mainly caused by the elongation of the tendinous tissue. It was suggested that the elasticity of VL tendinous tissue enabled VL fascicles to develop force at closer length to their optimal length and kept the maximum shortening velocity of VL fascicles low during slow pedaling.     

Age-related changes in contractile properties of single skeletal fibers from the soleus muscle.

Peak absolute force, specific tension (peak absolute force per cross-sectional area), cross-sectional area, maximal unloaded shortening velocity (Vo; determined by the slack test), and myosin heavy chain (MHC) isoform compositions were determined in 124 single skeletal fibers from the soleus muscle of 12-, 24-, 30-, 36-, and 37-mo-old Fischer 344 Brown Norway F1 Hybrid rats. All fibers expressed the type I MHC isoform. The mean Vo remained unchanged from 12 to 24 mo but did decrease significantly from the 24- to 30-mo time period (from 1.71 +/- 0.13 to 0.85 +/- 0.09 fiber lengths/s). Fiber cross-sectional area remained constant until 36 mo of age, at which time there was a 20% decrease from the values at 12 mo of age (from 5,558 +/- 232 to 4,339 +/- 280 micrometer2). A significant decrease in peak absolute force of single fibers occurred between 12 and 24 mo of age (from 51 +/- 2 x 10(-5) to 35 +/- 2 x 10(-5) N) and then remained constant until 36 mo, when another 43% decrease occurred. Like peak absolute force, the specific tension decreased significantly between 12 and 24 mo by 20%, and another 32% decline was observed at 37 mo. Thus, by 24 mo, there was a dissociation between the loss of fiber cross-sectional area and force. The results suggest time-specific changes of the contractile properties with aging that are independent of each other. Underlying mechanisms responsible for the time-dependent and contractile property-specific changes are unknown. Age-related changes in the molecular dynamics of myosin may be the underlying mechanism for altered force production. The presence of more than one beta/slow MHC isoform may be the mechanism for the altered Vo with age.     

Muscle dynamics: Dependence of muscle length on changes in external load

A phenomenological theory of muscle dynamics has been elaborated on the basis of data obtained in experiments on hind limb extensor muscles of narcotized cat. Functional dependence of muscle length on external load was explored in conditions of a constant frequency of the efferent stimulation. It was shown that the system under study could be presented for a rather wide class of input signals as a system with nonlinear statics and linear dynamics. The nonlinear statics was shown to be determined mainly by the hysteretical effects of muscle contraction, whereas dynamic element was described by the first order linear differential equation corresponding to the traditional three-component mechanical model of the muscle. A hypothesis was proposed to explain the hysteresis in active muscle on the basis of functioning of the troponin-tropomyosin regulatory complex. Elaborated mathematical model of muscle dynamics can be used to predict and evaluate changes in the muscle length evoked by arbitrary changes in the external load.     

Optical depolarization changes in single, skinned muscle fibers. Evidence for cross-bridge involvement.

Optical ellipsometry studies of single, skinned muscle fibers conducted on the diffraction orders have yielded spectra that are sensitive to the state of the fiber. The linearly polarized light field vector becomes elliptically polarized as it passes through the fiber and may be collected at the diffraction orders. Fibers that have been subjected to extraction of myosin (0.6 M KCl) retain a weak diffraction pattern and exhibit a substantially decreased depolarization of incident linearly polarized light. A significant decrease in polarization is seen in skinned fibers that are subject to an increase in pH from 7.0 to 8.0. This increase in pH results in a decrease of approximately 30% in the depolarization angle of single fibers. The major decrease in depolarization angle that we observe at pH 8.0 is consistent with the notion that as cross-bridges move out from the shaft of the thick filament, their ability to cause depolarization of the incident linearly polarized light decreases. This interpretation is also consistent with the work of Ueno and Harrington where the decrease in the ability to cross-link S-1 and S-2 to the thick filament at pH 8.2 suggests cross-bridge movement away from the thick filament. A large decrease in birefringence, seen after treatment of skinned fibers with alpha-chymotrypsin, appears to be related to the breakdown of myosin into rod, S-1, heavy meromyosin, and light meromyosin.     

Changes of myoplasmic calcium concentration during fatigue in single mouse muscle fibers

Measurements of the intracellular free concentration of Ca2+ ([Ca2+]i) were performed during fatiguing stimulation of intact, single muscle fibers, which were dissected from a mouse foot muscle and loaded with fura-2. Fatigue, which was produced by repeated 100-Hz tetani, generally occurred in three phases. Initially, tension declined rapidly to approximately 90% of the original tension (0.9 Po) and during this period the tetanic [Ca2+]i increased significantly (phase 1). Then followed a lengthy period of almost stable tension production and tetanic [Ca2+]i (phase 2). Finally, both the tetanic [Ca2+]i and tension fell relatively fast (phase 3). The resting [Ca2+]i rose continuously throughout the stimulation period. A 10-s rest period during phase 3 resulted in a significant increase of both tetanic [Ca2+]i and tension, whereas a 10-s pause during phase 2 did not have any marked effect. Application of caffeine under control conditions and early during phase 2 resulted in a substantial increase of the tetanic [Ca2+]i but no marked tension increase, whereas caffeine applied at the end of fatiguing stimulation (tension depressed to approximately 0.3 Po) gave a marked increase of both tetanic [Ca2+]i and tension. The tetanic [Ca2+]i for a given tension was generally higher during fatiguing stimulation than under control conditions. Fatigue developed more rapidly in fibers exposed to cyanide. In these fibers there was no increase of tetanic [Ca2+]i during phase 1 and the increase of the resting [Ca2+]i during fatiguing stimulation was markedly larger. The present results indicate that fatigue produced by repeated tetani is caused by a combination of reduced maximum tension-generating capacity, reduced myofibrillar Ca2+ sensitivity, and reduced Ca2+ release from the sarcoplasmic reticulum. The depression of maximum tension-generating capacity develops early during fatiguing stimulation and it is of greatest importance for the force decline at early stages of fatigue. As fatigue gets more severe, reduced Ca2+ sensitivity and reduced Ca2+ release become quantitatively more important for the tension decline.     

Local calcium changes regulate the length of growth cone filopodia

Previous studies have demonstrated that the free intracellular calcium concentration ([Ca(2+)](i)) in growth cones can act as an important regulator of growth cone behavior. Here we investigated whether there is a spatial and temporal correlation between [Ca(2+)](i) and one particular aspect of growth cone behavior, namely the regulation of growth cone filopodia. Calcium was released from the caged compound NP-EGTA (o-nitrophenyl EGTA tetrapotassium salt) to simulate a signaling event in the form of a transient increase in [Ca(2+)](i). In three different experimental paradigms, we released calcium either globally (within an entire growth cone), regionally (within a small area of the lamellipodium), or locally (within a single filopodium). We demonstrate that global photolysis of NP-EGTA in growth cones caused a transient increase in [Ca(2+)](i) throughout the growth cone and elicited subsequent filopodial elongation that was restricted to the stimulated growth cone. Pharmacological blockage of either calmodulin or the Ca(2+)-dependent phosphatase, calcineurin, inhibited the effect of uncaging calcium, suggesting that these enzymes are acting downstream of calcium. Regional uncaging of calcium in the lamellipodium caused a regional increase in [Ca(2+)](i), but induced filopodial elongation on the entire growth cone. Elevation of [Ca(2+)](i) locally within an individual filopodium resulted in the elongation of only the stimulated filopodium. These findings suggest that the effect of an elevation of [Ca(2+)](i) on filopodial behavior depends on the spatial distribution of the calcium signal. In particular, calcium signals within filopodia can cause filopodial length changes that are likely a first step towards directed filopodial steering events seen during pathfinding in vivo.     

Arsenazo III and antipyrylazo III calcium transients in single skeletal muscle fibers

The metallochrome calcium indicators arsenazo III and antipyrylazo III have been introduced individually into cut single frog skeletal muscle fibers from which calcium transients have been elicited either by action potential stimulation or by voltage-clamp pulses of up to 50 ms in duration. Calcium transients recorded with both dyes at selected wavelengths have similar characteristics when elicited by action potentials. Longer voltage-clamp pulse stimulation reveals differences in the late phases of the optical signals obtained with the two dyes. The effects of different tension blocking methods on Ca transients were compared experimentally. Internal application of EGTA at concentrations up to 3 mM was demonstrated to be efficient in blocking movement artifacts without affecting Ca transients. Higher EGTA concentrations affect the Ca signals' characteristics. Differential effects of internally applied EGTA on tension development as opposed to calcium transients suggest that diffusion with binding from Ca++ release sites to filament overlap sites may be significant. The spectral characteristics of the absorbance transients recorded with arsenazo III suggest that in situ recorded signals cannot be easily interpreted in terms of Ca concentration changes. A more exhaustic knowledge of the dye chemistry and/or in situ complications in the use of the dye will be necessary.     

The effect of external calcium and magnesium depletion on single nerve fibers

The three types of motor axons found in the walking legs of the lobster were shown to respond differently upon exposure to calcium-free solutions. While all fiber types became more excitable initially in calcium-free solutions, only openers became spontaneously active. Fast closers showed the least reduction in rheobase value upon calcium depletion. After 5 minutes in calcium-free solution all fibers showed a rise in rheobase value, and more rapid accommodation. A natural period for spontaneous firing of opener fibers was disclosed. Following such a spontaneous discharge, low amplitude rhythmical potentials were recorded. These small potentials had the same period as the spontaneous spikes. The role of calcium ion in the excitable process was discussed. Magnesium ion was shown to act synergistically with calcium ion. All fiber types became spontaneously active in solutions deprived of both calcium and magnesium. Subsequent hypoexcitability was more pronounced in calcium- and magnesium-depleted solutions than it was in only calcium-depleted solutions.     

Volume changes of the myosin lattice resulting from repetitive stimulation of single muscle fibers.

Single muscle fibers at 1 degreesC were subjected to brief tetani (20 Hz) at intervals of between 20 s and 300 s over a period of up to 2 h. A band lattice spacing increased during this period at a rate inversely dependent on the rest interval between tetani. Spacing increased rapidly during the first 10 tetani at a rate equivalent to the production of 0.04 mOsmol.liter-1 of osmolyte per contraction, then continued to expand at a much slower rate. For short rest intervals, where lattice expansion was largest, spacing increased to a limiting value between 46 and 47 nm (sarcomere length 2.2 micrometer), corresponding to accumulation of 30 mOsmol.liter-1 of osmolytes, where it remained constant until repetitive stimulation was terminated. At this limiting spacing, force was reduced by up to 30%. The effect of lattice swelling on the lattice compression that accompanies isometric force recovery from unloaded shortening was to increase the compression, similar to that observed in hypotonic media at a similar spacing. During recovery from repetitive stimulation, spacing recompressed to its original value with a half-time of 15-30 min. These findings suggest that mechanical activity produces an increase in osmotic pressure within the cell as a result of product accumulation from cross-bridge and sarcoplasmic reticulum ATPases and glycolysis.