Isometrically Contracting Research Articles

A New and Improved View of Force Production

A central question in muscle contraction is “How is the energy release in ATP hydrolysis converted into mechanical force and work?” With the elucidation of the actomyosin ATPase reaction in the 1970s (1,2) and the fact that a large amount of free energy is produced during Pi release from an actomyosin-ADP-Pi species (3), the Pi release step from actomyosin-ADP-Pi must be associated with the generation of force and shortening. It was also known that increases in the [Pi] concentration in isometrically contracting skinned muscle fibers reduced isometric force (4).

Stiffness and number of crossbridges attached in active frog muscle: a reply to Professor Lombardi

We do not share Professor Lombardi’s confidence that the crossbridge stiffness and the fraction of myosin heads (crossbridges) attached in an isometric contraction are experimentally well established for muscles in these two frog species (Lombardi 2011). In particular we are not persuaded that the crossbridge stiffness is high (*3 pN/ nm) and the fraction of attached heads is low (20% in Rana esculenta and 30% in Rana temporaria). Determination of the crossbridge stiffness in muscle requires knowledge of the fraction of heads attached in an isometric contraction, since it is the product of the crossbridge stiffness and this fraction that is usually determined by subtracting the filament compliance from the half-sarcomere compliance. This inter-relation would cause an overestimate of the crossbridge stiffness to lead to the fraction of crossbridges attached being underestimated. An alternative approach which avoids knowing the fraction of heads attached in an isometric contraction is to determine the crossbridge stiffness in the rigor state. Unfortunately this is subject to large errors because the half-sarcomere compliance in the rigor state is not much larger than the contribution of filament compliance. The thermodynamic approach used for Rana esculenta (Decostre et al. 2005), which appeared at first to offer the hope of determining the crossbridge stiffness directly, is regrettably without foundation as we showed in Appendix 2 of our review (Offer and Ranatunga 2010). The fraction of myosin heads attached in an isometric contraction can be obtained from the ratio of the contribution of crossbridges to the half-sarcomere compliance in rigor to that in the active state. But as the rigor crossbridge compliance contribution can be determined only with a large error, the fraction of heads attached in an isometric contraction also carries a large error. In an earlier study (Linari et al. 1998) thick filament compliance in Rana esculenta was estimated from changes in the spacing of the M3 myosin meridional X-ray reflection. From the ratio of crossbridge compliance contributions in active and rigor muscle, the fraction of heads in isometric contraction may be calculated as 0.43 ± 0.087. But deciding that the M6 reflection should give a more reliable indication of the thick filament compliance, required their estimate of the fraction of heads attached in an isometric contraction to fall to 0.20 ± 0.12 (Linari et al. 2007). Even these large standard errors do not include the errors in determining the thick filament compliance. Moreover, there may be a systematic error in this quantity since it is by no means clear that the M6 reflection is exclusively contributed by detached heads. In our review we discussed the wide range of values that have been reported for the fraction of myosin heads attached (Offer and Ranatunga 2010). In particular, Bershitsky et al. (1997) gauged the number of heads attached in a stereospecific manner in isometrically contracting semitendinosus fibres of Rana temporaria from the intensity of the first actin layer line. At 5–6 C 27% were stereospecifically attached, but after rapidly raising the temperature to 16–19 C by a T-jump, this rose to 46%. This implied that the total fraction of heads attached was more than 46%. Because of this uncertainty, in the main body of the review we were careful to express crossbridge G. Offer K. W. Ranatunga (&) School of Physiology & Pharmacology, The Medical Sciences Building, University of Bristol, Bristol, UK e-mail: k.w.ranatunga@bristol.ac.uk

Electron Tomography of Cryofixed, Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

BackgroundIsometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ.MethodologyWe used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the “target zone”, situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening.ConclusionWe present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments.

Probing myosin structural conformation in vivo by second-harmonic generation microscopy

Understanding of complex biological processes requires knowledge of molecular structures and measurement of their dynamics in vivo. The collective chemomechanical action of myosin molecules (the molecular motors) in the muscle sarcomere represents a paradigmatic example in this respect. Here, we describe a label-free imaging method sensitive to protein conformation in vivo. We employed the order-based contrast enhancement by second-harmonic generation (SHG) for the functional imaging of muscle cells. We found that SHG polarization anisotropy (SPA) measurements report on the structural state of the actomyosin motors, with significant sensitivity to the conformation of myosin. In fact, each physiological/biochemical state we probed (relaxed, rigor, isometric contraction) produced a distinct value of polarization anisotropy. Employing a full reconstruction of the contributing elementary SHG emitters in the actomyosin motor array at atomic scale, we provide a molecular interpretation of the SPA measurements in terms of myosin conformations. We applied this method to the discrimination between attached and detached myosin heads in an isometrically contracting intact fiber. Our observations indicate that isometrically contracting muscle sustains its tetanic force by steady-state commitment of 30% of myosin heads. Applying SPA and molecular structure modeling to the imaging of unstained living tissues provides the basis for a generation of imaging and diagnostic tools capable of probing molecular structures and dynamics in vivo.

Is Slower Myosin Cross-Bridge Kinetics in Tg-D166V Preparations Due to Decreased Myosin RLC Phosphorylation?

In this report we have investigated a particularly malignant phenotype of Familial Hypertrophic Cardiomyopathy (FHC) associated with the 166 Aspartic Acid to Valine (D166V) mutation in the ventricular myosin regulatory light chain (RLC). We show that the rates of myosin cross-bridge attachment and dissociation are significantly different in isometrically contracting cardiac myofibrils from transgenic (Tg)-D166V compared to Tg-WT mice. A single molecule approach was taken where the fluorescence anisotropy of rhodamine phalloidin labeled actin protomers was measured in cardiac myofibrils undergoing isometric contraction. Orientation of an actin molecule oscillated between two states, corresponding to the actin-bound and actin-free states of the myosin cross-bridge. The rates of cross-bridge attachment as well as cross-bridge dissociation were significantly decreased in isometrically contracting Tg-D166V myofibrils (binding, 1.4 s−1; detachment, 1.2 s−1) compared to Tg-WT myofibrils (binding, 3 s−1; detachment, 1.3 s−1). The duty ratio of the cross-bridge cycle, equal to the fraction of the total cycle time that cross-bridge remains attached to actin, was 47% inTg-D166V myofibrils and 30% in Tg-WT. Immunoblotting of cardiac myofibrils used for kinetics studies demonstrated a large reduction in RLC phosphorylation in Tg-D166V vs. Tg-WT myofibrils. These data are in accord with our previous findings in skinned and intact papillary muscles showing slower fiber kinetics and prolonged force transients in Tg-D166V fibers compared to Tg-WT preparations (Kerrick et al., FASEB J. 23: 855, 2009). Similarly, the level of RLC phosphorylation in muscle extracts from Tg-D166V ventricles was decreased. Our cellular and single molecule data suggest that a mutation-dependent decrease in RLC phosphorylationcould initiate the slower kinetics of the D166V cross-bridgesand ultimately lead to abnormal cardiac muscle contraction. Supported by NIH-HL071778 (DSC), NIH-AR048622 (JB) and NIH-HL090786 (to JB and DSC).

Electron Tomography of Cryofixed, Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

We have applied multivariate data analysis to 38.7 nm repeat segments obtained from electron tomograms of isometrically contracting insect flight muscle fibers, mechanically monitored, rapidly frozen, freeze-substituted and thin sectioned. Improved resolution reveals for the first time the helix of F-actin subunits in the thin filament with sufficient clarity that an atomic model can be built into the density independent of the myosin cross-bridges, thereby providing an objective method for identifying weak and strong actin-myosin attachments. The tomogram shows strong binding myosin attachments on only four F-actin subunits midway between successive troponin complexes; these actin subunits comprise the “target zone” of active contraction. Improved quantitation facilitates a more detailed description of weak and strong myosin attachments all along the thin filament including for the first time myosin heads contacting the thin filament on troponin. Most strong binding actin attachments consist of single myosin heads but 28% of bound heads are 2-headed myosin attachments. Strong binding attachments show an axial lever arm range of 77° sweeping out a distance of 12.9 nm. The azimuthal range for the lever arm of strong binding attachments is 127° with a distribution nearly completely to one side of the initial crystallographic structures used for the fitting. There is no apparent coupling between axial angle, representing progress through the power stroke of myosin, and the azimuthal lever arm angle. Two types of weak actin attachments are observed. One type, which is found exclusively on target zone actin subunits, appears to represent prepowerstroke intermediates. The other, which appears to have a different function, is positioned on the M-ward side of the target zone, i.e. the direction toward which filaments slide during sarcomere shortening. Its motor domain contacts tropomyosin rather than actin. Supported by NIH.

Single molecule kinetics in the familial hypertrophic cardiomyopathy D166V mutant mouse heart

One of the sarcomeric mutations associated with a malignant phenotype of familial hypertrophic cardiomyopathy (FHC) is the D166V point mutation in the ventricular myosin regulatory light chain (RLC) encoded by the MYL2 gene. In this report we show that the rates of myosin cross-bridge attachment and dissociation are significantly different in isometrically contracting cardiac myofibrils from right ventricles of transgenic (Tg)-D166V and Tg-WT mice. We have derived the myosin cross-bridge kinetic rates by tracking the orientation of a fluorescently labeled single actin molecule. Orientation (measured by polarized fluorescence) oscillated between two states, corresponding to the actin-bound and actin-free states of the myosin cross-bridge. The rate of cross-bridge attachment during isometric contraction decreased from 3 s−1 in myofibrils from Tg-WT to 1.4 s−1 in myofibrils from Tg-D166V. The rate of detachment decreased from 1.3 s−1 (Tg-WT) to 1.2 s−1 (Tg-D166V). We also showed that the level of RLC phosphorylation was largely decreased in Tg-D166V myofibrils compared to Tg-WT. Our findings suggest that alterations in the myosin cross-bridge kinetics brought about by the D166V mutation in RLC might be responsible for the compromised function of the mutated hearts and lead to their inability to efficiently pump blood.

Rotation of actin monomers during isometric contraction of skeletal muscle

Cyclic interactions of myosin and actin are responsible for contraction of muscle. It is not self-evident, however, that the mechanical cycle occurs during steady-state isometric contraction where no work is produced. Studying cross-bridge dynamics during isometric steady-state contraction requires an equilibrium time-resolved method (not involving application of a transient). This work introduces such a method, which analyzes fluctuations of anisotropy of a few actin molecules in muscle. Fluorescence anisotropy, indicating orientation of an actin protomer, is collected from a volume of a few attoliters (10(-18) L) by confocal total internal reflection (CTIR) microscopy. In this method, the detection volume is made shallow by TIR illumination, and narrow by confocal aperture inserted in the conjugate image plane. The signal is contributed by approximately 12 labeled actin molecules. Shortening of a myofibril during contraction is prevented by light cross-linking with 1-ethyl-3-[3-dimethylamino)-propyl]-carbodiimide. The root mean-squared anisotropy fluctuations are greater in isometrically contracting than in rigor myofibrils. The results support the view that during isometric contraction, cross-bridges undergo a mechanical cycle.

Role of the right dorsal premotor cortex in “physiological” mirror EMG activity

A distributed cortical network enables the lateralization of intended unimanual movements, i.e., the transformation from a default mirror movement to a unimanual movement. Little is known about the exact functional organization of this "non-mirror transformation" network. Involvement of the right dorsal premotor cortex (dPMC) was suggested because its virtual lesion by high-frequency repetitive transcranial magnetic stimulation (rTMS) increased the excitability of the left primary motor cortex (M1) during unilateral isometric contraction of a left hand muscle (Cincotta et al., Neurosci Lett 367: 189-93, 2004). However, no behavioural effects were observed in that experimental protocol. Here we tested behaviourally twelve healthy volunteers to find out whether focal disruption of the right dPMC by "off-line" One Hz rTMS (900 pulses, 115% of resting motor threshold) enhances "physiological" mirroring. This was measured by an established protocol (Mayston et al., Ann Neurol 45: 583-94, 1999) that quantifies the mirror increase in the electromyographic (EMG) level in the isometrically contracting abductor pollicis brevis (APB) muscle of one hand during brief phasic contractions performed with the APB of the other hand. Mirroring in the right APB significantly increased after real rTMS of the right dPMC. In contrast, no change in mirroring was seen with sham rTMS of the right dPMC, real rTMS of the right M1, or real rTMS of the left dPMC. These findings strongly support the hypothesis that the right dPMC is part of the non-mirror transformation cortical network.

Mapping of movement in the isometrically contracting human soleus muscle reveals details of its structural and functional complexity.

It is becoming increasingly apparent that precise knowledge of the anatomic features of muscle, aponeurosis, and tendons is necessary for understanding how a muscle-tendon complex generates force and accomplishes length changes. This report presents both anatomic and functional data from the human soleus muscle acquired by using magnetic resonance imaging. The results show a strong relationship between the complex three-dimensional structure of the muscle-tendon system and the intramuscular distribution of tissue velocities during in vivo isometric contractions. The proximal region of the muscle is unipennate, whereas the midregion has a radially bipennate hemicylindrical structure, and the distal region is quadripennate. Tissue velocity mapping shows that the highest velocity regions overlay the aponeuroses connected to the Achilles tendon. These are located on the anterior and posterior surfaces of the muscle. The lowest velocities overlay the aponeuroses connected to the origin of the muscle and are generally located intramuscularly.

Finite element model of intramuscular pressure during isometric contraction of skeletal muscle

The measurement of in vivo intramuscular pressure (IMP) has recently become practical and IMP appears well correlated with muscle tension. A numerical model of skeletal muscle was developed to examine the mechanisms producing IMP. Unipennate muscle is modelled as a two-dimensional material continuum that is incompressible and nonlinearly anisotropic. The finite element technique is used to calculate IMP and muscle stress during passive stretch and during isometric contraction. A novel element models the contractile portion of muscle, incorporating sarcomere length–force and velocity–force relations. A range of unipennate muscle geometries can be modelled.The model was configured to simulate the rabbit tibialis anterior muscle over a range of lengths. Simulated IMP and stress results were validated against animal experimentation data. The simulation agreed well with the experimental data over the range of 0.8–1.1 of the optimal length. Severe pressure gradients were produced near the musculo-tendinous junctions while IMP was more uniform in the central muscle belly. IMP and muscle stress in relaxed (unstimulated) muscle increased nonlinearly with muscle length. IMP and stress in isometrically contracting muscle showed a local maximum at optimal length and were reduced at shorter lengths. At muscle lengths longer than optimal, stress and IMP increased predominately due to tension in the passive elastic structures.

Evidence that phosphate release is the rate-limiting step on the overall ATPase of psoas myofibrils prevented from shortening by chemical cross-linking.

It has been suggested that the mechanical condition determines the rate-limiting step of the ATPase of the myosin heads in fibers: when fibers are isometrically contracting, the ADP release kinetics are rate-limiting, but as the strain is reduced and the fibers are allowed to shorten, the ADP release kinetics accelerate and P(i) release becomes rate-limiting. We have put this idea to the test with myofibrils as a model because with these both mechanical and chemical kinetic measurements are possible. With relaxed or rapidly shortening myofibrils, P(i) release is rate-limiting and (A)M.ADP.P(i) states accumulate in the steady state [Lionne, C., et al. (1995) FEBS Lett. 364, 59]. We have now studied the kinetics of P(i) release with chemically cross-linked myofibrils that, when adequately cross-linked, appear to be a good model for isometric contraction. By using a method that is specific for free P(i) and rapid quench flow that measures the amount of (A)M.ADP.P(i) states and free P(i), we show that (A)M.ADP.P(i) states predominate which suggests that the overall ATPase is limited by P(i) release kinetics. Therefore, under our experimental conditions with myofibrils prevented from shortening, the concentration of (A)M.ADP states is low, as with rapidly shortening and relaxed myofibrils. This result is difficult to reconcile with the sensitivity of force development in fibers and myofibrils to P(i) which implies interaction of P(i) with an (A)M.ADP state. We discuss two models for accommodating the mechanical and chemical kinetics with reference to the duty cycle in skeletal muscle.

Structural Features of Cross-Bridges in Isometrically Contracting Skeletal Muscle

Two-dimensional x-ray diffraction was used to investigate structural features of cross-bridges that generate force in isometrically contracting skeletal muscle. Diffraction patterns were recorded from arrays of single, chemically skinned rabbit psoas muscle fibers during isometric force generation, under relaxation, and in rigor. In isometric contraction, a rather prominent intensification of the actin layer lines at 5.9 and 5.1 nm and of the first actin layer line at 37 nm was found compared with those under relaxing conditions. Surprisingly, during isometric contraction, the intensity profile of the 5.9-nm actin layer line was shifted toward the meridian, but the resulting intensity profile was different from that observed in rigor. We particularly addressed the question whether the differences seen between rigor and active contraction might be due to a rigor-like configuration of both myosin heads in the absence of nucleotide (rigor), whereas during active contraction only one head of each myosin molecule is in a rigor-like configuration and the second head is weakly bound. To investigate this question, we created different mixtures of weak binding myosin heads and rigor-like actomyosin complexes by titrating MgATP γS at saturating [Ca 2+] into arrays of single muscle fibers. The resulting diffraction patterns were different in several respects from patterns recorded under isometric contraction, particularly in the intensity distribution along the 5.9-nm actin layer line. This result indicates that cross-bridges present during isometric force generation are not simply a mixture of weakly bound and single-headed rigor-like complexes but are rather distinctly different from the rigor-like cross-bridge. Experiments with myosin-S1 and truncated S1 (motor domain) support the idea that for a force generating cross-bridge, disorder due to elastic distortion might involve a larger part of the myosin head than for a nucleotide free, rigor cross-bridge.

Myosin head orientation and mobility during isometric contraction: effects of osmotic compression

We have correlated the mobility and the generation of force of myosin heads by applying radial compression to isometrically contracting muscle fibers. Osmotic pressure was produced by dextran T-500, and its effect on the orientation and mobility of myosin heads labeled with N-(1-oxy-2,2,5,5-tetramethyl-4-pyperidinyl)maleimide was observed by conventional and saturation-transfer electron paramagnetic resonance methods. A biphasic behavior is spectral changes coinciding with the tension dependence was observed as the fibers were compressed. At diameters above the equilibrium spacing, the large myosin head disorder characteristic during contraction in the absence of compression was largely maintained, whereas the mobility decreased threefold, from tauR approximately 25 microseconds to approximately 80-90 microseconds. The inhibition of fast microsecond motions was not accompanied by tension loss, implying that these motions are not necessary for force generation. At diameters below the equilibrium spacing, both the disorder and the mobility decreased dramatically in parallel with the tension inhibition, suggesting that slower microsecond motions and the disorder of the myosin head are necessary for muscle function.

The descending limb of the force‐sarcomere length relation of the frog revisited.

1. We studied the descending limb of the force-sarcomere length relation in single frog muscle fibres using sarcomere isometric contractions. 2. Sarcomere length was measured simultaneously with two independent methods: a laser diffraction method and a segment length method that detects the distance between two markers attached to the surface of the fibre, about 800 microns apart. Both methods were used to keep sarcomeres at constant length during contraction. 3. Fibres were selected for low resting tension since it was known from previous experiments that for such fibres the force developed by fixed-end tetani is much higher than that predicted by the degree of filament overlap. 4. With fixed-end tetani, force decline with increase of sarcomere length was small between 2.0 and 3.0 microns. At a sarcomere length of 3.0 microns, force was about 90% of maximal. 5. With sarcomere isometric tetani, force was considerably lower than with fixed-end tetani. Force was maximal at about 2.1 microns and decreased to zero at about 3.6 microns. At intermediate lengths the descending limb was within 80 nm of the values predicted from filament overlap. 6. We investigated why force of fixed-end contractions was much higher than that generated by sarcomere isometric contractions. 7. During the force plateau of fixed-end tetani at sarcomere lengths longer than about 2.0-2.2 microns, sarcomeres in the fibres mid-region were not isometric, but instead stretched slowly. By measuring the force-velocity relation it was shown that this slow stretch elevates active force well beyond sarcomere isometric force. 8. Stretch of the central region was also observed during the tetanic force rise. This was shown to result in an increase of passive force that grew larger at longer sarcomere lengths. At about 3.6 microns the increase of passive force was similar to the total force generated by fixed-end contractions at this length. 9. Laser diffraction and segment length methods gave the same results, diminishing the chance that any systematic artifact underlies our findings. 10. While earlier experiments from this laboratory carried out on fibres held at constant length during contraction did not reveal a linear descending limb, the present results support the linear descending limb as a characteristic feature of isometrically contracting sarcomeres.

Distribution of cross-bridges in isometrically contracting muscle: its deduction from the Huxley-Simmons theory and the effect of filament elasticity.

The author attempted to deduce a possible cross-bridge (XB) distribution as a function of x, the amount of stretch of the XB, by applying the Huxley-Simmons theory with their recent experimental data to a steady isometric contraction. I found no XB distribution which satisfied at once the Huxley-Simmons theory, their data and observations in muscle energetics and biochemistry. Further, a computation with a simple model suggested that the XB distribution could be broadened by the elasticity of the thick and/or the thin filament if it existed.

Ultrasonic Velocity Fluctuation in Muscle

Ultrasonic velocity fluctuations were measured in frog sartorius muscle during isometric contraction and relaxation by the time-to-amplitude conversion method. Spectrum analysis of the fluctuations indicated that the velocity fluctuation increases in isometrically contracting muscle in the frequency range of 10–30 Hz. Acoustic emission from muscle was also searched for in connection with the velocity fluctuations.

Angle of active site of myosin heads in contracting muscle during sudden length changes.

The change in orientation of myosin crossbridges in contracting muscle during sudden length changes was examined by fluorescence polarization. This study used a fluorescent ATP analogue, 1,N6-etheno-2-aza-ATP(epsilon-2-aza-ATP) as a probe. Its fluorescence is considerably enhanced upon binding with myosin and is dependent on the chemical state of the myosin-nucleotide complex in muscle. The results showed that nucleotides bound to crossbridges in the intermediate attached state (presumably AM-epsilon-2-aza-ADP-Pi) during isometric contraction are highly oriented at the same angle as that of AM in rigor with bound epsilon-2-aza-ADP. Furthermore the orientation of nucleotides bound to crossbridges in the attached state is not altered during sudden changes in length of isometrically contracting muscle. The results of this time-resolved measurement support the conclusion obtained from a previous steady-state experiment that change in axial orientation of the active site of the myosin head is not involved in force generation.

Mechanical properties of toad slow muscle attributed to non-uniform sarcomere lengths.

Tension changes have been measured during shortening or stretching movements applied to actively contracting motor units of the tonus bundle of the iliofibularis muscle of the toad Bufo marinus. During a slow, constant-velocity release tension fell, initially rapidly and then more slowly. The size of the fall, particularly later in the movement, depended on a number of factors including the duration of the isometric contraction before the onset of shortening, the amount of tension developed by the motor unit and the length of the muscle. When an isometrically contracting motor unit was rapidly shortened, the rate of rise on re-development of tension following the release was significantly slower than at the onset of the contraction. This effect was more marked if the release was preceded by a longer period of isometric contraction, if the experiment was carried out at shorter muscle lengths or if a smaller motor unit was used. If, following a period of isometric contraction, stimulation was interrupted, and a release-stretch movement applied to quickly bring the level of force down to near zero, and then stimulation recommenced , the final level of re-developed tension was less than that immediately before the release. The size of the tension deficit following re-development was larger for small motor units and at short muscle lengths. When the duration of the contraction before release was increased the size of the deficit also increased. A deficit could be prevented if the muscle was allowed to relax passively before the shortening movement was commenced. Stretch of actively contracting slow muscle produced an initial steep tension rise followed at times by a transient fall before tension slowly rose again. The transient fall became larger at short muscle lengths, and after long-duration contractions before stretch. Its tension dependence was less easy to establish because of complications involving changes in the relative series compliance. All of the above observations could be accounted for by an explanation based on the development of sarcomere non- uniformities in slow muscle fibres, produced as a result of non-uniform activation of the fibre membrane through the distributed nerve supply.

Quasi-elastic light-scattering studies of single skeletal muscle fibers

Measurements were made of the intensity autocorrelation function, g(2)[tau], of light scattered from intact frog muscle fibers. During the tension plateau of an isometric tenanus, scattered field statistics were approximately Gaussian and intensity fluctuations were quasi-stationary. The half time, tau 1/2, for the decay of g(2)[tau] was typically 70 ms at a scattering angle of 30 degrees. The decay rate, 1/tau 1/2, of g(2)[tau] varied roughly linearly with the projection of the scattering vector on the fiber axis. 1/tau 1/2 was greater during the tension creep phase of tetani of highly stretched fibers, but was roughly independent of sarcomere length during the tension plateau. g(2)[tau] measured during rest or on diffraction pattern maxima during isometric contraction were flat with low amplitudes. These results are consistent with a model of a 200-mu m segment of an isometrically contracting fiber in which scattering material possesses relative axial velocities of 1-2 mu m/s accompanied by relative axial displacements greater than 0.1 mu m. The slow (1-2 mu m/s) motion of one portion of the fiber relative to another observed under the microscope (500X) during isometric contraction is consistent with the light-scattering results. Structural fluctuations on the scale of the myofibrillar sarcomere which may arise from asynchronous cycling of cross-bridges must involve relative axial velocities less than 3 mu m/s or relative axial displacements less than 0.05 mu m.