A-band shortening in single fibers of frog skeletal muscle.

The question of whether A-bands shorten during contraction was investigated using two methods: high-resolution polarization microscopy and electron microscopy. During shortening from extended sarcomere lengths in the passive state, sarcomere-length changes were essentially accounted for by I-band shortening. During active shortening under otherwise identical conditions, the sarcomere length change was taken up approximately equally by A- and I-bands. Several potential artifacts that could give rise to apparent A-band shortening were considered and judged unlikely. Results obtained with polarization microscopy were similar to those obtained with electron microscopy. Thus, modest but significant thick filament shortening appears to occur during active sarcomere shortening under physiological conditions.     

ULTRASTRUCTURE OF THE RESTING AND CONTRACTED STRIATED MUSCLE FIBER AT DIFFERENT DEGREES OF STRETCH

Passive stretch, isometric contraction, and shortening were studied in electron micrographs of striated, non-glycerinated frog muscle fibers. The artifacts due to the different steps of preparation were evaluated by comparing sarcomere length and fiber diameter before, during, and after fixation and after sectioning. Tension and length were recorded in the resting and contracted fiber before and during fixation. The I filaments could be traced to enter the A band between the A filaments on both sides of the I band, creating a zone of overlap which decreased linearly with stretch and increased with shortening. This is consistent with a sliding filament model. The decrease in the length of the A and I filaments during isometric contraction and the finding that fibers stretched to a sarcomere length of 3.7 µ still developed 30 per cent of the maximum tetanic tension could not be explained in terms of the sliding filament model. Shortening of the sarcomeres near the myotendinous junctions which still have overlap could account for only one-sixth of this tension, indicating that even those sarcomeres stretched to such a degree that there is a gap between A and I filaments are activated during isometric contraction (increase in stiffness). Shortening, too, was associated with changes in filament length. The diameter of A filaments remained unaltered with stretch and with isometric contraction. Shortening of 50 per cent was associated with a 13 per cent increase in A filament diameter. The area occupied by the fibrils and by the interfibrillar space increased with shortening, indicating a 20 per cent reduction in the volume of the fibrils when shortening amounted to 40 per cent.     

Titin-induced force enhancement and force depression: A ‘sticky-spring’ mechanism in muscle contractions?

The sliding filament and crossbridge theories do not suffice to explain a number of muscle experiments. For example, from the entire muscle to myofibrils, predictions of these theories were shown to underestimate the force output during and after active tissue stretch. The converse applies to active tissue shortening.In addition to the crossbridge cycle, we propose that another molecular mechanism is effective in sarcomere force generation. We suggest that, when due to activation, myosin binding sites are available on actin, the giant protein titin's PEVK region attaches itself to the actin filament at those sites. As a result, the molecular spring length is dramatically reduced. This leads to increased passive force when the sarcomere is stretched and to decreased or even negative passive force when the sarcomere shortens. Moreover, during shortening, the proposed mechanism interferes with active-force production by inhibiting crossbridges.Incorporation of a simple ‘sticky-spring’ mechanism model into a Hill-type model of sarcomere dynamics offers explanations for several force-enhancement and force-depression effects. For example, the increase of the sarcomere force compared to the force predicted solely by the sliding filament and crossbridge theories depends on the stretch amplitude and on the working range. The same applies to the decrease of sarcomere force during and after shortening. Using only literature data for its parameterization, the model predicts forces similar to experimental results.     

CONTRACTILITY AND ULTRASTRUCTURE IN GLYCEROL-EXTRACTED MUSCLE FIBERS : I. The Relationship of Contractility to Sarcomere Length

This study was undertaken to determine whether glycerol-extracted rabbit psoas muscle fibers can develop tension and shorten after being stretched to such a length that the primary and secondary filaments no longer overlap. A method was devised to measure the initial sarcomere length and the ATP-induced isotonic shortening in prestretched isolated fibers subjected to a small preload (0.02 to 0.15 P₀). At all degrees of stretch, the fiber was able to shorten (60 to 75 per cent): to a sarcomere length of 0.7 µ when the initial length was 3.7 µ or less, and to an increasing length of 0.9 to 1.8 µ with increasing initial sarcomere length (3.8 to 4.4 µ). At sarcomere lengths of 3.8 to 4.5 µ, overlap of filaments was lost, as verified by electron microscopy. The variation in sarcomere length within individual fibers has been assessed by both light and electron microscopic measurements. In fibers up to 10 mm in length the stretch was evenly distributed along the fiber, and with sarcomere spacings greater than 4 µ there was only a slight chance of finding sarcomeres with filament overlap. These observations are in apparent contradiction to the assumption that an overlap of A and I filaments is necessary for tension generation and shortening.     

Polarized light observations on striated muscle contraction in a mite

Contraction of individual sarcomeres within the living mite Tarsonemus sp. was observed by polarized light microscopy. In unflattened animals the usual range of contraction was such that the minimum sarcomere length approximated the length of the A region, and the maximum sarcomere length was about twice the length of the A region. The central sarcomeres of the dorsal metapodosomal muscles were observed in detail. The A band length increased slightly with increasing sarcomere length since the regression of I region length on sarcomere length had an average slope of 0.91. When the A band length in a sarcomere which was shortening was compared with the length when the same sarcomere lengthened, no significant difference was seen. The A band of each sarcomere seemed to act as a not too rigid limit to further shortening; this agreed with the reversible shortening of a muscle in which the A band had been experimentally shortened. An H region was visible at long sarcomere lengths and was not visible at short sarcomere lengths, even when the muscle was actively shortening. The rate of change of H region length with sarcomere length suggested that I filament length may increase as sarcomere length increases. Despite this effect and the small increase in A length with sarcomere length, the results are considered to be consistent with a model in which shortening occurs by the relative movement of A and I filaments, with little or no change in length of either set of filaments. Sarcomere shortening was clearly associated with an increase in the retardation of the A region.     

IMMUNOHISTOCHEMICAL LOCALIZATION OF CONTRACTILE PROTEINS IN LIMULUS STRIATED MUSCLE

Limulus paramyosin and myosin were localized in the A bands of glycerinated Limulus striated muscle by the indirect horseradish peroxidase-labeled antibody and direct and indirect fluorescent antibody techniques. Localization of each protein in the A band varied with sarcomere length. Antiparamyosin was bound at the lateral margins of the A bands in long (~ 10.0 µ) and intermediate (~ 7.0 µ) length sarcomeres, and also in a thin line in the central A bands of sarcomeres, 7.0–~6.0 µ. Antiparamyosin stained the entire A bands of short sarcomeres (<6.0). Conversely, antimyosin stained the entire A bands of long sarcomeres, showed decreased intensity of central A band staining except for a thin medial line in intermediate length sarcomeres, and was bound only in the lateral A bands of short sarcomeres. These results are consistent with a model in which paramyosin comprises the core of the thick filament and myosin forms a cortex. Differential staining observed using antiparamyosin and antimyosin at various sarcomere lengths and changes in A band lengths reflect the extent of thick-thin filament interaction and conformational change in the thick filament during sarcomeric shortening.     

The stiffness of skeletal muscle in isometric contraction and rigor: the fraction of myosin heads bound to actin.

Step changes in length (between -3 and +5 nm per half-sarcomere) were imposed on isolated muscle fibers at the plateau of an isometric tetanus (tension T0) and on the same fibers in rigor after permeabilization of the sarcolemma, to determine stiffness of the half-sarcomere in the two conditions. To identify the contribution of actin filaments to the total half-sarcomere compliance (C), measurements were made at sarcomere lengths between 2.00 and 2.15 microm, where the number of myosin cross-bridges in the region of overlap between the myosin filament and the actin filament remains constant, and only the length of the nonoverlapped region of the actin filament changes with sarcomere length. At 2.1 microm sarcomere length, C was 3.9 nm T0(-1) in active isometric contraction and 2.6 nm T0(-1) in rigor. The actin filament compliance, estimated from the slope of the relation between C and sarcomere length, was 2.3 nm microm(-1) T0(-1). Recent x-ray diffraction experiments suggest that the myosin filament compliance is 1.3 nm microm(-1) T0(-1). With these values for filament compliance, the difference in half-sarcomere compliance between isometric contraction and rigor indicates that the fraction of myosin cross-bridges attached to actin in isometric contraction is not larger than 0.43, assuming that cross-bridge elasticity is the same in isometric contraction and rigor.     

THE RELATIONSHIP BETWEEN MYOFILAMENT PACKING DENSITY AND SARCOMERE LENGTH IN FROG STRIATED MUSCLE

In cross-sections of single fibers from the frog semitendinosus muscle the number of thick myofilaments per unit area (packing density) is a direct function of the sarcomere length. Our data, derived from electron microscopic studies, fit well with other data derived from in vivo, low-angle X-ray diffraction studies of whole semitendinosus muscles. The data are consistent with the assumption that the sarcomere of a fibril maintains a constant volume during changes in sarcomere length. The myofilament lattice, therefore, expands as the sarcomere shortens. Since the distance between adjacent myofilaments is an inverse square root function of sarcomere length, the interaction of the thick and the thin myofilaments during sarcomere shortening may occur over distances which increase 70 A or more. The "expanding-sarcomere, sliding-filament" model of sarcomere shortening is discussed in terms of the current concepts of muscle architecture and contraction.     

Electron Microscopic Studies of Skeletal and Cardiac Muscle of a Benthic Fish. I. Myofibrillar Structure in Resting and Contracted Muscle

SYNOPSIS. Electron microscopic studies are reported on glycerinatedskeletal and cardiac muscle of a benthic fish, Coryphaenoidesspecies. In white skeletal muscle, the sarcomeres have a restinglength of approximately 1.8 µ, with thick filaments 1.4µ and thin filaments 0.75 µ in length. These dimensionsare somewhat shorter than filament lengths of oilier vertebratemuscles, possibly due to the elfect of volume increase duringassembly of thick and thin filaments at high hydrostatic pressure.During ATP-induced contraction of Coryphaenoides muscle fromsarcomere lengths of 1.8 µ to 1.6 µ, there is acharacteristic interdigitation of thick and thin filaments,with decrease in I band length and no change in length of thickor thin filaments. However, in sarcomeres contracted to lengthsof 1.5 µ. to 1.2 µ, there is a slight shorteningof the A band, apparently due to shortening of thick filaments,that occurs despite the presence of residual I band in the samesarcomeres. There is no obvious crumpling or distortion of thickfilaments during contraction to sarcomere lengths as low as1.0 µ, but filament organization undergoes extensive disarrayat sarcomere lengths approaching 0.7 µ. Although effectsfrom heterogeneity of filament length cannot be excluded withcertainty, the present evidence does suggest that contractionot Coryphaenoides muscle from 1.6 µ to 1.0 µ sarcomerelengih is accompanied by shortening of thick filaments consequentto a structural change within the thick filament core.     

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.     

Overextended sarcomeres regain filament overlap following stretch

Sarcomere overextension has been widely implicated in stretch-induced muscle injury. Yet, sarcomere overextensions are typically inferred based on indirect evidence obtained in muscle and fibre preparations, where individual sarcomeres cannot be observed during dynamic contractions. Therefore, it remains unclear whether sarcomere overextensions are permanent following injury-inducing stretch-shortening cycles, and thus, if they can explain stretch-induced force loss. We tested the hypothesis that overextended sarcomeres can regain filament overlap in isolated myofibrils from rabbit psoas muscles. Maximally activated myofibrils (n=13) were stretched from an average sarcomere length of 2.6±0.04μm by 0.9μm sarcomere(-1) at a speed of 0.1μm sarcomere(-1)s(-1) and immediately returned to the starting lengths at the same speed (sarcomere strain=34.1±2.3%). Myofibrils were then allowed to contract isometrically at the starting lengths (2.6μm) for ～30s before relaxing. Force and individual sarcomere lengths were measured continuously. Out of the 182 sarcomeres, 35 sarcomeres were overextended at the peak of stretch, out of which 26 regained filament overlap in the shortening phase while 9 (～5%) remained overextended. About 35% of the sarcomeres with initial lengths on the descending limb of the force-length relationship and ～2% of the sarcomeres with shorter initial lengths were overextended. These findings provide first ever direct evidence that overextended sarcomeres can regain filament overlap in the shortening phase following stretch, and that the likelihood of overextension is higher for sarcomeres residing initially on the descending limb.     

Interaction between series compliance and sarcomere kinetics determines internal sarcomere shortening during fixed-end contraction

The interaction between contractile force and in-series compliance was investigated for the intact skeletal muscle-tendon unit (MTU) of Rana pipiens semitendinosus muscles during fixed-end contraction. It was hypothesized that internal sarcomere shortening is a function of the length-force characteristics of contractile and series elastic components. The MTUs (n=18) were dissected, and, while submerged in Ringer's solution, muscles were activated at nine muscle lengths (-2 to +6 mm relative to optimal length in 1 mm intervals), while measuring muscle force and sarcomere length (SL) by laser diffraction. The MTU was clamped either at the bone (n=6), or at the proximal and distal ends of the aponeuroses (n=6). Muscle fibers were also trimmed along with aponeuroses down to 5-20 fibers and identical measurements were performed (n=6). The magnitude of shortening decreased as MTU length increased. The magnitude of shortening ranged from -0.08 to 0.3 microm, and there was no significant difference between delta SL as a function of clamp location. When aponeuroses were trimmed, sarcomere shortening was not observed at L(0) and longer. These results suggest that the aponeurosis is the major contributor to in-series compliance. Results also support our hypothesis but there also appear to be other factors affecting internal sarcomere shortening. The functional consequence of internal sarcomere shortening as a function of sarcomere length was to skew the muscle length-tension relationship to longer sarcomere lengths.     

Changes in thick filament length in Limulus striated muscle

Here we describe the change in thick filament length in striated muscle of Limulus, the horseshoe crab. Long thick filaments (4.0 microns) are isolated from living, unstimulated Limulus striated muscle while those isolated from either electrically or K+-stimulated fibers are significantly shorter (3.1 microns) (P less than 0.001). Filaments isolated from muscle glycerinated at long sarcomere lengths are long (4.4 microns) while those isolated from muscle glycerinated at short sarcomere lengths are short (2.9 microns) and the difference is significant (P less than 0.001). Thin filaments are 2.4 microns in length. The shortening of thick filaments is related to the wide range of sarcomere lengths exhibited by Limulus telson striated muscle.     

Knockdown of fast skeletal myosin-binding protein C in zebrafish results in a severe skeletal myopathy

Myosin-binding protein C (MyBPC) in the muscle sarcomere interacts with several contractile and structural proteins. Mutations in the cardiac isoform (MyBPC-3) in humans, or animal knockout, are associated with cardiomyopathy. Function of the fast skeletal isoform (MyBPC-2) in living muscles is less understood. This question was addressed using zebrafish models, combining gene expression data with functional analysis of contractility and small-angle x-ray diffraction measurements of filament structure. Fast skeletal MyBPC-2B, the major isoform, was knocked down by >50% using morpholino antisense nucleotides. These morphants exhibited a skeletal myopathy with elevated apoptosis and up-regulation of factors associated with muscle protein degradation. Morphant muscles had shorter sarcomeres with a broader length distribution, shorter actin filaments, and a wider interfilament spacing compared with controls, suggesting that fast skeletal MyBPC has a role in sarcomere assembly. Active force was reduced more than expected from the decrease in muscle size, suggesting that MyBPC-2 is required for optimal force generation at the cross-bridge level. The maximal shortening velocity was significantly increased in the MyBPC-2 morphants, but when related to the sarcomere length, the difference was smaller, reflecting that the decrease in MyBPC-2B content and the resulting myopathy were accompanied by only a minor influence on filament shortening kinetics. In the controls, equatorial patterns from small-angle x-ray scattering revealed that comparatively few cross-bridges are attached (as evaluated by the intensity ratio of the 11 and 10 equatorial reflections) during active contraction. X-ray scattering data from relaxed and contracting morphants were not significantly different from those in controls. However, the increase in the 11:10 intensity ratio in rigor was lower compared with that in controls, possibly reflecting effects of MyBPC on the cross-bridge interactions. In conclusion, lack of MyBPC-2 results in a severe skeletal myopathy with structural changes and muscle weakness.     

Ultrastructure of muscle fibres in head and axial muscles of the perch (Perca fluviatilis L.)

Summary White, pink, red and deep red fibres, selected from a head muscle and from axial muscles of the perch, show significant differences in actin filament length, Z line thickness, Z line lattice space, myofibril girth, the percentages volume occupied by T system and terminal cisternae of the SR, and in the degree of T system SR contact per sarcomere. In both muscles the degree of T system SR contact decreases in the order: white, pink, red, deep red, which suggests a decrease of contraction velocity in the same order.The position of the T system (at the Z line or at the AI junction) is related to the actin filament length. The actin filaments in the red fibres are appreciably longer than in the white, which suggests that the sarcomeres of the red fibres have a broader length-tension curve. The Z line thickness is positively correlated with the actin filament length and, in the white and the red fibres, negatively with the degree of sarcomere shortening. Thicker Z lines are suggested to allow greater sarcomere sizes (length or girth).The percentage volume occupied by mitochondria varies independently of the extent of membrane systems.The ultrastructural characteristics of the fibre types are in agreement with the functional roles as reported in literature.     

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)     

Three-dimensional reconstruction of a simple Z-band in fish muscle

The three-dimensional structure of the Z-band in fish white muscle has been investigated by electron microscopy. This Z-band is described as simple, since in longitudinal sections it has the appearance of a single zigzag pattern connecting the ends of actin filaments of opposite polarity from adjacent sarcomeres. The reconstruction shows two pairs of links, the Z-links, between one actin filament and the facing four actin filaments in the adjacent sarcomere. The members of each pair have nearly diametrically opposed origins. In relation to one actin filament, one pair of links appears to bind along the final 10 nm of the actin filament (proximal site) and the other pair binds along a region extending from 5 to 20 nm from the filament end (distal site). Between one pair and the other, there is a rotation of approximately 80 degrees round the filament axis. A Z-link with a proximal site at the end of one actin filament attaches at a distal site on the oppositely oriented actin filaments of the facing sarcomere and vice versa. The length of each Z-link is consistent with the length of an alpha-actinin molecule. An additional set of links located 10-15 nm from the center of the Z-band occurs between actin filaments of the same polarity. These polar links connect the actin filaments along the same direction on each side of the Z-band. The three-dimensional structure appears to have twofold screw symmetry about the central plane of the Z-band. Only approximate twofold rotational symmetry is observed in directions parallel to the actin filaments. Previous models of the Z-band in which four identical and rotationally symmetrical links emanate from the end of one actin filament and span across to the ends of four actin filaments in the adjacent sarcomere are therefore incorrect.     

CONTRACTILITY AND ULTRASTRUCTURE IN GLYCEROL-EXTRACTED MUSCLE FIBERS : II. Ultrastructure in Resting and Shortened Fibers

Glycerol-extracted rabbit psoas muscle fibers were examined by electron microscopy both before and after ATP-induced isotonic shortening. Ultrastructural changes were correlated with the initial sarcomere length and the degree of shortening. The ultrastructural appearance of the resting fiber at rest length was identical with that described by H. E. Huxley and Hanson. At sarcomere lengths greater than 3.7 to 3.8 µ, the A and I filaments were detached and separated by a gap. The presence of "gap" filaments was confirmed, and evidence is presented which indicates that these filaments form connections between the ends of the A and I filaments. Shortening from initial sarcomere lengths at which the filaments overlapped took place through sliding of the filaments. If shortening was initiated from sarcomere lengths at which there was a gap, a narrowing of the I band was brought about by a curling of the I filaments at the boundary between the A and I bands. No evidence could be found that the I filaments moved into the A band.     

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.