Ultrastructure of skeletal muscle fibers studied by a plunge quick freezing method: myofilament lengths.

We have set up a system to rapidly freeze muscle fibers during contraction to investigate by electron microscopy the ultrastructure of active muscles. Glycerinated fiber bundles of rabbit psoas muscles were frozen in conditions of rigor, relaxation, isometric contraction, and active shortening. Freezing was carried out by plunging the bundles into liquid ethane. The frozen bundles were then freeze-substituted, plastic-embedded, and sectioned for electron microscopic observation. X-ray diffraction patterns of the embedded bundles and optical diffraction patterns of the micrographs resemble the x-ray diffraction patterns of unfixed muscles, showing the ability of the method to preserve the muscle ultrastructure. In the optical diffraction patterns layer lines up to 1/5.9 nm-1 were observed. Using this method we have investigated the myofilament lengths and concluded that there are no major changes in length in either the actin or the myosin filaments under any of the conditions explored.     

Why Muscle is an Efficient Shock Absorber

Skeletal muscles power body movement by converting free energy of ATP hydrolysis into mechanical work. During the landing phase of running or jumping some activated skeletal muscles are subjected to stretch. Upon stretch they absorb body energy quickly and effectively thus protecting joints and bones from impact damage. This is achieved because during lengthening, skeletal muscle bears higher force and has higher instantaneous stiffness than during isometric contraction, and yet consumes very little ATP. We wish to understand how the actomyosin molecules change their structure and interaction to implement these physiologically useful mechanical and thermodynamical properties. We monitored changes in the low angle x-ray diffraction pattern of rabbit skeletal muscle fibers during ramp stretch compared to those during isometric contraction at physiological temperature using synchrotron radiation. The intensities of the off-meridional layer lines and fine interference structure of the meridional M3 myosin x-ray reflection were resolved. Mechanical and structural data show that upon stretch the fraction of actin-bound myosin heads is higher than during isometric contraction. On the other hand, the intensities of the actin layer lines are lower than during isometric contraction. Taken together, these results suggest that during stretch, a significant fraction of actin-bound heads is bound non-stereo-specifically, i.e. they are disordered azimuthally although stiff axially. As the strong or stereo-specific myosin binding to actin is necessary for actin activation of the myosin ATPase, this finding explains the low metabolic cost of energy absorption by muscle during the landing phase of locomotion.     

Fine structure in near-field and far-field laser diffraction patterns from skeletal muscle fibers.

Regions of muscle fibers that are many sarcomeres in length and uniform with regard to striation spacing, curvature, and tilt have been observed by light microscopy. We have investigated the possibility that these sarcomere domains can explain the fine structure in optical diffraction patterns of skeletal muscle fibers. We studied near-field and far-field diffraction patterns with respect to fiber translation and to masking of the laser beam. The position of diffracted light in the near-field pattern depends on sarcomere length and position of the diffracting regions within the laser beam. When a muscle fiber was translated longitudinally through a fixed laser beam, the fine structural lines in the near-field diffraction pattern moved in the same direction and by the same amount as the fiber movement. Translation of the muscle fiber did not result in fine structure movement in the far-field pattern. As the laser beam was incrementally masked from one side, some fine structural lines in both the near-field and far-field diffraction patterns changed in intensity while others remained the same. Eventually, all the fine structural lines broadened and decreased in intensity. Often a fine structural line increased in intensity or a dark area in the diffraction pattern became brighter as the laser beam was restricted. From these results we conclude that the fine structure in the laser diffraction pattern is due to localized and relatively uniform regions of sarcomeres (domains) and to cross interference among light rays scattered by different domains.     

Decrease in light diffraction intensity of contracting muscle fibres

Single fibres from the semitendinosus muscle of frog were illuminated normally with a He–Ne laser. The intensity transient and fine structure pattern of light diffracted from the fibre undergoing isometric twitches were measured. During fibre shortening, the intensity decreased rapidly and the fine structure pattern preserved its shape and moved swiftly away from the undiffracted laser beam. The fine structure patterns of the contracting and resting fibre were nearly identical. The ratio of intensities of the contracting and resting fibre of the same sarcomere length was determined as a function of the time elapsed after fibre stimulation. The time-resolved intensity ratio increased with sarcomere length and became unity when sarcomere length was between 3.5 m and 3.7 m. A diffraction theory based on the sarcomere unit was developed. It contained a parameter describing the strength of filament interaction. The comparison between the theory and data shows that the initial intensity drop during contraction is primarily due to filament interactions. At a later stage of contraction, sarcomere disorder becomes the major component causing the intensity to decrease. Diffraction models which use the Debye-Waller formalism to explain the intensity decrease are discussed. The sarcomere-unit diffraction model is applied to previously reported intensity measurements from active fibres.     

Direct modeling of x-ray diffraction pattern from skeletal muscle in rigor

Available high-resolution structures of F-actin, myosin subfragment 1 (S1), and their complex, actin-S1, were used to calculate a 2D x-ray diffraction pattern from skeletal muscle in rigor. Actin sites occupied by myosin heads were chosen using a "principle of minimal elastic distortion energy" so that the 3D actin labeling pattern in the A-band of a sarcomere was determined by a single parameter. Computer calculations demonstrate that the total off-meridional intensity of a layer line does not depend on disorder of the filament lattice. The intensity of the first actin layer A1 line is independent of tilting of the "lever arm" region of the myosin heads. Myosin-based modulation of actin labeling pattern leads not only to the appearance of the myosin and "beating" actin-myosin layer lines in rigor diffraction patterns, but also to changes in the intensities of some actin layer lines compared to random labeling. Results of the modeling were compared to experimental data obtained from small bundles of rabbit muscle fibers. A good fit of the data was obtained without recourse to global parameter search. The approach developed here provides a background for quantitative interpretation of the x-ray diffraction data from contracting muscle and understanding structural changes underlying muscle contraction.     

Evidence for structurally different attached states of myosin cross-bridges on actin during contraction of fish muscle.

Using data from fast time-resolved x-ray diffraction experiments on the synchrotrons at Daresbury and (Deutsches Elektronen Synchrotron [DESY]), it is shown that during contraction of fish muscle there are at least two distinct configurations of myosin cross-bridges on actin, that they appear to have different tension producing properties and that they probably differ in the axial tilt of the cross-bridges on actin. Evidence is presented for newly observed myosin-based layer lines in patterns from active fish muscle, together with intensity changes of the actin layer lines. On the equator, the 110 reflection changes much faster (time for 50% change t1/2 = 21 +/- 4 ms after activation) than the 100 reflection (t1/2 = 35 +/- 8 ms) and tension (t1/2 = 41 +/- 3 ms) during the rising phase of tetanic contractions. These and higher order reflections have been used to show the time course of mass attachment at actin during this rising phase. Mass arrival (t1/2 = 25 ms) precedes tension by approximately 15 ms. Analysis has been carried out to evaluate the effects of changes in sarcomere length during the tetanus. It is shown that any such effects are very small. Difference "equatorial" electron density maps between active muscle at a time when mass arrival at actin is just complete, but the tension is still rising, and at a later time well into the tension plateau, show that the structural difference between the lower and higher force states corresponds to mass movement consistent with axial swinging of heads from a nonstereospecific actin attached state (low force) to a more stereospecific (high force) state.     

X-ray diffraction evidence for the extensibility of actin and myosin filaments during muscle contraction.

To clarify the extensibility of thin actin and thick myosin filaments in muscle, we examined the spacings of actin and myosin filament-based reflections in x-ray diffraction patterns at high resolution during isometric contraction of frog skeletal muscles and steady lengthening of the active muscles using synchrotron radiation as an intense x-ray source and a storage phosphor plate as a high sensitivity, high resolution area detector. Spacing of the actin meridional reflection at approximately 1/2.7 nm-1, which corresponds to the axial rise per actin subunit in the thin filament, increased about 0.25% during isometric contraction of muscles at full overlap length of thick and thin filaments. The changes in muscles stretched to approximately half overlap of the filaments, when they were scaled linearly up to the full isometric tension, gave an increase of approximately 0.3%. Conversely, the spacing decreased by approximately 0.1% upon activation of muscles at nonoverlap length. Slow stretching of a contracting muscle increased tension and increased this spacing over the isometric contraction value. Scaled up to a 100% tension increase, this corresponds to a approximately 0.26% additional change, consistent with that of the initial isometric contraction. Taken together, the extensibility of the actin filament amounts to 3-4 nm of elongation when a muscle switches from relaxation to maximum isometric contraction. Axial spacings of the layer-line reflections at approximately 1/5.1 nm-1 and approximately 1/5.9 nm-1 corresponding to the pitches of the right- and left-handed genetic helices of the actin filament, showed similar changes to that of the meridional reflection during isometric contraction of muscles at full overlap. The spacing changes of these reflections, which also depend on the mechanical load on the muscle, indicate that elongation is accompanied by slight changes of the actin helical structure possibly because of the axial force exerted by the actomyosin cross-bridges. Additional small spacing changes of the myosin meridional reflections during length changes applied to contracting muscles represented an increase of approximately 0.26% (scaled up to a 100% tension increase) in the myosin periodicity, suggesting that such spacing changes correspond to a tension-related extension of the myosin filaments. Elongation of the myosin filament backbone amounts to approximately 2.1 nm per half sarcomere. The results indicate that a large part (approximately 70%) of the sarcomere compliance of an active muscle is caused by the extensibility of the actin and myosin filaments; 42% of the compliance resides in the actin filaments, and 27% of it is in the myosin filaments.     

X-ray diffraction indicates that active cross-bridges bind to actin target zones in insect flight muscle.

We report the first time-resolved study of the two-dimensional x-ray diffraction pattern during active contraction in insect flight muscle (IFM). Activation of demembranated Lethocerus IFM was triggered by 1.5-2.5% step stretches (risetime 10 ms; held for 1.5 s) giving delayed active tension that peaked at 100-200 ms. Bundles of 8-12 fibers were stretch-activated on SRS synchrotron x-ray beamline 16.1, and time-resolved changes in diffraction were monitored with a SRS 2-D multiwire detector. As active tension rose, the 14.5- and 7.2-nm meridionals fell, the first row line dropped at the 38.7 nm layer line while gaining a new peak at 19.3 nm, and three outer peaks on the 38.7-nm layer line rose. The first row line changes suggest restricted binding of active myosin heads to the helically preferred region in each actin target zone, where, in rigor, two-headed lead bridges bind, midway between troponin bulges that repeat every 38.7 nm. Halving this troponin repeat by binding of single active heads explains the intensity rise at 19.3 nm being coupled to a loss at 38.7 nm. The meridional changes signal movement of at least 30% of all myosin heads away from their axially ordered positions on the myosin helix. The 38.7- and 19.3-nm layer line changes signal stereoselective attachment of 7-23% of the myosin heads to the actin helix, although with too little ordering at 6-nm resolution to affect the 5.9-nm actin layer line. We conclude that stretch-activated tension of IFM is produced by cross-bridges that bind to rigor's lead-bridge target zones, comprising < or = 1/3 of the 75-80% that attach in rigor.     

Wavelet analysis of the m. vastus lateralis surface electromyogram activity in incremental tests until exhaustion using bicycle and knee extension exercises

Continuous wavelet analysis was used to study the spectral characteristics of the m. vastus lateralis electromyographic activity in two I : rhythmic knee-joint extensions and cycling. Wavelet analysis of surface electromyograms (EMGs) recorded during cyclic contractions of the same muscle during the two types of movements showed differences in the pattern of changes in the timefrequency characteristics of the signal during a single cycle of movements with different loads, as well as differences in the slow variations of spectral characteristics related to the development of muscle fatigue during the tests. It was shown that, during cycling at low loads (the beginning of the test) the EMG activity peaked during the second half of the muscle contraction (the angle in the knee joint was ≈140°); the increase in the load at the end of the test led to a shift of the peak to the beginning of the active phase of movement, while the median frequency of the instant wavelet spectra during the muscle contraction remained almost unchanged. During knee-joint extension, the maximum EMG activity was observed at the very end of the active phase of movement for all loads, and the median frequency significantly increased with increasing angle at the knee joint. The long-term time course of the EMG intensity growth during these tests also differed, whereas the changes in the wavelet-spectrum median frequencies were practically the same: they increased during both tests.     

Thin filament elasticity and its role in the muscle contraction

The available experimental methods do not allow one to establish unambiguously the molecular structural events during muscle contraction. To resolve the existing controversies, I have devised an unconventional original computer program. The new approach allows the reconstruction of the hexagonal lattice of the sarcomere for different muscle states and verification of the structure by comparison of the calculated Fourier spectra with the real diffraction patterns. Previously, by the use of this approach, the real structure of a myosin filament from vertebrate striated muscle has been reconstructed (http://zope.ibib.waw.pl/pspk). In this work, a reconstruction for the thin filament is presented for three states: relaxed, after activation, and during contraction. Good consistency of the calculated Fourier spectra with the real diffraction patterns available in the literature suggests that the thin filament, due to flexibility, plays an active part in muscle contraction, as myosin cross-bridges do.     

Theory of light diffraction by single skeletal muscle fibers.

A theoretical discussion is presented describing the diffraction of laser light by a single fiber of striated muscle. The complete three-dimensional geometry of the fiber has been taken into consideration. The basic repeated unit is taken as the sarcomere of a single myofibril, including its cylindrical geometry. The single fiber is considered as the sum of myofibrils up to the fiber dimensions. When proper phasing is taken into account, three cases of interest are analyzed. (a) When the adjacent myofibrils are totally aligned with respect to their index of refraction regions (e.g., A and I bands), then the diffraction pattern reflects that of a larger striated cylinder with the dimensions of the fiber. (b) When a particular skew plane develops for the myofibril elements, additional Bragg reflection occurs at certain specific sarcomere lengths, and intensity asymmetry amongst the diffracted orders occurs. (c) When the myofibril phasing changes in a random fashion, while all sarcomeres remain at the same length, then intensity decrease is directly related to the phase deviation from a reference phase point. This condition may well describe a fiber undergoing active isometric contraction.     

Quantification of the dynamic properties of EMG patterns during gait.

A technique for analyzing and comparing the dynamic properties of electromyographic (EMG) patterns collected during gait is presented. A gait metric is computed, consisting of both magnitude (amplitude) and phase (timing) components. For the magnitude component, the processed EMG pattern is compared to a normative EMG pattern obtained under similar walking conditions, where the metric is incremented if the muscle is firing during expected active regions or is silent during expected inactive regions. The magnitude metric is penalized when the EMG is silent during phases of expected activity or when the EMG is active in regions of expected inactivity. The phase component of the metric computes the percentage of the gait cycle when the muscle is firing appropriately, that is, active in expected active regions and silent in expected inactive regions. The magnitude and phase components of the metric are normalized and combined to yield the EMG pattern that demonstrates the closest characteristics compared to normative gait data collected under similar walking conditions. Using experimental data, the proposed gait metric was tested and accurately reflects the observed changes in the EMG patterns. Clinical uses for the gait metric are discussed in relation to gait therapies, such as determining optimal gait training conditions in individuals following stroke and spinal cord injury.     

Ultrastructure of a molluscan smooth muscle during phasic contractions and in the relaxed state

Tension and X-ray diffraction patterns are not always correlated in the smooth anterior retractor muscle (ABRM) of Mytilus edulis. The muscle produces equatorial intensity profiles of X-ray diffraction patterns corresponding to either a relaxed or a contracted structure. During phasic contractions, comprising a contracted as well a a relaxed phase, the diffracted intensity on the equator at 0.003 A^?1 changes within the first 10s after onset of stimulation. The tension reaches a maximum after about the same time. The time dependence of this intensity change during phasic contraction has been measured. It shows that the tension decays within 10s, but the relaxed structure needs 30–40 s to reestablish. There is no difference between the observed intensities from the tonic and phasic contracted states. Inactivated muscles with minimum tension, normally termed relaxed, can have either a “contracted” or a relaxed structure.     

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 MgATPgammaS at saturating [Ca2+] 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.     

Thin filament flexibility and its role in muscle contraction

Contemporary experimental methods do not allow unequivocal determination of molecular structural events during muscle contraction. To analyze existing contradictions, an original computer program has been developed. This program reconstructs the hexagonal lattice of a sarcomere for different states of muscle and finds the most realistic structure by comparing the calculated Fourier spectrum with the actual diffraction pattern. Previously, the new approach allowed reconstructing the actual structure of a myosin filament from mammalian striated muscle (http://zope.ibib.waw.pl/pspk). In this work, the thin filament is reconstructed for three states: relaxed, activated, and contracting. The good fit between the calculated Fourier spectra and the actual diffraction patterns taken from the literature suggests that the thin filament owing to its flexibility may play an active role in muscle contraction, as myosin cross-bridges do.     

Variation of muscle stiffness with force at increasing speeds of shortening

Single frog skeletal muscle fibers were attached to a servo motor and force transducer by knotting the tendons to pieces of wire at the fiber insertions. Small amplitude, high frequency sinusoidal length changes were then applied during tetani while fibers contracted both isometrically and isotonically at various constant velocities. The amplitude of the resulting force oscillation provides a relative measure of muscle stiffness. It is shown from an analysis of the transient force responses observed after sudden changes in muscle length applied both at full and reduced overlap and during the rising phase of short tetani that these responses can be explained on the basis of varying numbers of cross bridges attached at the time of the length step. Therefore, the stiffness measured by the high frequency length oscillation method is taken to be directly proportional to the number of cross bridges attached to thin filament sites. It is found that muscle stiffness measured in this way falls with increasing shortening velocity, but not as rapidly as the force. The results suggest that at the maximum velocity of shortening, when the external force is zero, muscle stiffness is still substantial. The findings are interpreted in terms of a specific model for muscle contraction in which the maximum velocity of shortening under zero external load arises when a force balance is attained between attached cross bridges some of which are aiding and others opposing shortening. Other interpretations of these results are also discussed.     

Theoretical Fraunhofer light diffraction patterns calculated from three-dimensional sarcomere arrays imaged from isolated cardiac cells at rest.

Sarcomere striation positions have been obtained throughout the volumes of calcium-tolerant resting heart cells by direct computer interfaced high-resolution optical imaging. Each sarcomere position is stored in a three-dimensional (3-D) matrix array from which Fraunhofer light diffraction patterns have been calculated using numerical methods based on Fourier transforms. Diffraction patterns have been calculated from heart cell data arrays oriented normal to a theoretical laser beam. Twelve characteristic features have been identified and described from these diffraction patterns that correlate to diffraction phenomena observed from both cardiac and skeletal muscle. This numerical approach provides the means to directly assess diffraction pattern formulation, the precision of layer line angular separation, layer-line intensity and angular asymmetries, line widths and fine structures in terms of the known diffracting source structures. These results confirm that theoretical calculations can predict real muscle diffraction patterns and their asymmetries.     

Studies on the structure of muscle. III. Phase contrast and electron microscopy of dipteran flight muscle

1. The flight muscles of blowflies are easily dispersed in appropriate media to form suspensions of myofibrils which are highly suitable for phase contrast observation of the band changes associated with ATP-induced contraction. 2. Fresh myofibrils show a simple band pattern in which the A substance is uniformly distributed throughout the sarcomere, while the pattern characteristic of glycerinated material is identical with that generally regarded as typical of relaxed vertebrate myofibrils (A, I, H, Z, and M bands present). 3. Unrestrained myofibrils of both fresh and glycerinated muscle shorten by not more than about 20 per cent on exposure to ATP. In both cases the A substance migrates during contraction and accumulates in dense bands in the Z region, while material also accumulates in the M region. It is proposed that these dense contraction bands be designated the C(z), and C(m) bands respectively. In restrained myofibrils, the I band does not disappear, but the C(z) and C(m) bands still appear in the presence of ATP. 4. The birefringence of the myofibrils decreases somewhat during contraction, but the shift of A substance does not result in an increase of birefringence in the C(z) and C(m) bands. It seems therefore that the A substance, if it is oriented parallel with the fibre axis in the relaxed myofibril, must exist in a coiled or folded configuration in the C hands of contracted myofibrils. 5. The fine structure of the flight muscle has been determined from electron microscopic examination of ultrathin sections. The myofibrils are of roughly hexagonal cross-section and consist of a regular single hexagonal array of compound myofilaments the cores of which extend continuously throughout all bands of the sarcomere in all states of contraction or relaxation so far investigated. 6. Each myofilament is joined laterally with its six nearest neighbours by thin filamentous bridges which repeat at regular intervals along the fibre axis and are present in the A, I, and Z, but not in the H or M bands. When stained with PTA, the myofilaments display a compound structure. In the A band, a lightly staining medullary region about 40 A in diameter is surrounded by a densely staining cortex, the over-all diameter of the myofilament being about 120 A. This thick cortex is absent in the I and H bands, but a thinner cortex is often visible. 7. It is suggested that the basic structure is a longitudinally continuous framework of F actin filaments, which are linked periodically by the lateral bridges (possibly tropomyosin). The A substance is free under certain conditions to migrate to the Z bands to form the C(z) bands. The material forming the C(m) bands possibly represents another component of the A substance. The results do not clearly indicate whether myosin is confined to the A bands or distributed throughout the sarcomere.     

Effect of activation by calcium on the x-ray diffraction pattern from insect flight muscle.

A low-angle X-ray diffraction pattern of calcium-activated Lethocerus flight muscle was formed and the intensities of various parts of the pattern observed by means of a proportional counter. The muscle was sinusoidally oscillated in length to produce mechanical work. The resultant changes in diffraction intensity were related to the state of the muscle and to the phase of the mechanical oscillatory cycle. The measurements were interpreted in terms of a movement of the heads of the myosin molecules into contact with the actin filaments. In these terms the results showed that between 10 and 20% of the myosin heads attached to actin during work-producing oscillation of the muscle. The time-course of this attachment followed that of tension generation with a small delay. Calculation suggests that not all of the myosin molecules attached to actin at any one moment were generating tension.