Changes in thick filament structure during compression of the filament lattice in relaxed frog sartorius muscle

Summary Equatorial X-ray diffraction patterns from relaxed, chemically-skinned frog sartorius muscles under a range of external osmotic pressures from 0 to 290 torr have been analysed and compared to the pattern from the relaxed intact muscle. Lattice spacings and electron density diagrams were determined as a function of external pressure. Reflection intensities, averaged over small ranges of pressures, were determined out to the 4,0 reflection; phases for the first five orders were established as + +––+ over the whole pressure range. As external pressure was increased, lattice spacing decreased, as did full width at half maximum density for both thick and thin filaments. Most of the lattice spacing and thick filament compression occurred at low pressure, whereas thin filaments were compressed proportionally to pressure over the whole pressure range. These conclusions were confirmed by fitting cylindrical models for filament density to the X-ray diffraction patterns. Axially-projected electron density across the A-band filament lattice showed that in relaxed muscle the thick filament projections (myosin heads) are concentrated in regions between adjacent thick filaments, as far as possible from the thin filaments, and they tend to become pushed against the thick filament backbone as the lattice is compressed. Both thick and thin filament axes can be displaced randomly from their lattice positions; on average this displacement is about twice as great for the thin filaments, accounting for their larger projected size as compared to isolated thin filaments and for their apparent decrease in diameter as the lattice is compressed.     

Actin filament mechanics in the laser trap

Numerous biological processes, including muscular contraction, depend upon the mechanical properties of actin filaments. One such property is resistance to bending (flexural rigidity, EI). To estimate EI, we attached the ends of fluorescently labelled actin filaments to two microsphere‘handles’ captured in independent laser traps. The positions of the traps were manipulated to apply a range of tensions (0--8 pN)to the filaments via the microsphere handles. With increasing filament tension, the displacement of the microspheres was inconsistent with a microsphere-filament system that is rigid. We maintain that this inconsistency is due to the microspheres rotating in the trap and the filaments bending near their attachments to accommodate this rotation. Fitting the experimental data to a simple model of this phenomena, we estimate actin's EI to be ×15 × 103 pN nm2, a value within the range of previously reported results, albeit using a novel method. These results both: support the idea that actin filaments are more compliant than historically assumed; and, indicate that without appropriately pretensioning the actin filament in similar laser traps, measurements of unitary molecular events (e.g. myosin displacement) may be significantly underestimated This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of sarcomere length on step size in relaxed rabbit psoas muscle

Recent experiments have shown that shortening and stretching of sarcomeres in single activated and unactivated myofibrils occur in stepwise fashion (Yang et al. (1998) Biophys J 74: 1473-1483; Blyakhman et al. (2001) Biophys J 81: 1093-1100; Yakovenko et al. (2002) Am J Physiol Cell Physiol 283: 735-742). Here, we carried out measurements on single myofibrils from rabbit psoas muscle to investigate steps in unactivated specimens in more detail. Activated and unactivated myofibrils were released and stretched in ramp-like fashion. The time course of length change in the single sarcomere was consistently stepwise. We found that in the unactivated myofibrils, step size depended on initial sarcomere length, diminishing progressively with increase of initial sarcomere length, whereas in the case of activated sarcomeres, step size was consistently 2.7 nm.     

The chicken muscle thick filament: temperature and the relaxed cross-bridge arrangement

Summary Although chicken myosin S1 has recently been crystallized and its structure analysed, the relaxed periodic arrangement of myosin heads on the chicken thick filament has not been determined. We report here that the cross-bridge array of chicken filaments is temperature sensitive, and the myosin heads become disordered at temperatures near 4° C. At 25° C, however, thick filaments from chicken pectoralis muscle can be isolated with a well ordered, near-helical, arrangement of cross-bridges as seen in negatively stained preparations. This periodicity is confirmed by optical diffraction and computed transforms of images of the filaments. These show a strong series of layer lines near the orders of a 43 nm near-helical periodicity as expected from X-ray diffraction. Both analysis of phases on the first layer line, and computer filtered images of the filaments, are consistent with a three-stranded arrangement of the myosin heads on the filament.     

On the role of the filament length distribution in the mechanics of semiflexible networks

This paper explores the effects of filament length polydispersity on the mechanical properties of semiflexible crosslinked polymer networks. Extending previous studies on monodisperse networks, we compute numerically the response of crosslinked networks of elastic filaments of bimodal and exponential length distributions. These polydisperse networks are subject to the same affine to nonaffine (A/NA) transition observed previously for monodisperse networks, wherein the decreases in either crosslink density or bending stiffness lead to a shift from affine, stretching-dominated deformations to nonaffine, bending-dominated deformations. We find that the onset of this transition is generally more sensitive to changes in the density of longer filaments than shorter filaments, meaning that longer filaments have greater mechanical efficiency. Moreover, in polydisperse networks, mixtures of long and short filaments interact cooperatively to generally produce a nonaffine mechanical response closer to the affine prediction than comparable monodisperse networks of either long or short filaments. Accordingly, the mechanical affinity of polydisperse networks is dependent on the filament length composition. Overall, length polydispersity has the effect of sharpening and shifting the A/NA transition to lower network densities. We discuss the implications of these results on experimental observation of the A/NA transition, and on the design of advanced materials.     

Interpretation of the X-ray diffraction pattern from relaxed skeletal muscle and modelling of the thick filament structure

Summary The first part of this paper is devoted to the model-building studies of our high resolution meridional X-ray diffraction patterns (in the region from 1/500 to 1/50 Å^–1) obtained from relaxed frog muscle. A one-dimensional model of thick filament was proposed which basically consists of two symmetrical arrays of 50 crossbridge crown projections. In the proximate and central zones of the filament the crossbridge crowns are regularly shifted with a 429 Å period and appear as triplets with a 130 Å distance between crowns, while the crowns in the distal parts of filament are regularly ordered with a 143 Å repeat. The centre-to-centre distance between regions with crossbridge perturbations is 7050 Å. The length of each crown projection is about 125 Å. The model includes also (1) C-protein component represented in each half of the filament by seven stripes of about 350 Å long and located 429 Å apart, (2) a uniform density of filament backbone of about 1.5 m length, and (3) 13 high density stripes in a central zone located with 223 Å period. The final model explains very well the positions and intensities of the main meridional reflections. A three-dimensional model of crossbridge configuration is described in the second part of the work. The model was constructed by using the intensity profiles of the first six myosin layer lines of the X-ray pattern from stretched muscle and taking into account the crossbridge perturbations and the axial size of crossbridge crown obtained from the one-dimensional studies. It was found that both myosin heads are tilted in opposite directions along the filament and wrap around the filament backbone. This crossbridge configuration corresponds well to the findings of Haselgrove. To take into account interference effects between thick filaments we proposed a model of the filament hexagonal lattice with disorder of the second kind. The cross-sectional width of crystalline domains in stretched muscle was estimated to be about 0.11 m. The models presented may be helpful in the interpretation of the X-ray diffraction patterns from contracting muscle.     

On the mechanics of the actin filament: the linear relationship between stiffness and yield strength allows estimation of the yield strength of thin filament in vivo

Comparison of the behaviour of actin filaments either modified with tetramethylrhodamine iodoacetamide or decorated with tetramethylrhodamine-phalloidin or with tropomyosin or with myosin subfragment 1 shows that, in all the cases, yield strength is linearly related to stiffness.     

Nonuniformity of sarcomere shortenings in the isolated rat ventricular myocyte

By projecting the image of a single ventricular myocyte upon a linear image sensor, the striation pattern was analyzed during resting and contracting states. During rest, the individual cycle length (ICL) of the striation pattern varied from 1.8 to 2.0 microm within a given cell. ICL measured every 2 ms fluctuated approximately 0.05 microm around the mean. The variance of temporal fluctuations was decreased by chelating the extracellular Ca(2+) and increased by the Ca(2+) overload. Blocking the Ca(2+) release channels with 10 micromol/l ryanodine reversed this increase. In the power spectral density, an increase in the power occurred in the frequency range below 10 Hz. This increase should reflect overall kinetics of both intracellular Ca(2+) handling and responses of contractile filaments, because the same pattern was observed when spontaneous contractions occurred as well as when contractions were evoked by activating the L-type Ca(2+) channels. It is suggested that the temporal fluctuations of ICL in the resting state are caused by spontaneous Ca(2+) release from ryanodine receptors on the sarcoplasmic reticulum. Furthermore, under evoked contractions the shortenings of ICL were spatially inhomogeneous. These findings of nonuniform sarcomere shortenings are consistent with the temporal and spatial inhomogeneity of Ca(2+) transients.     

Muscle sarcomere length following passive jaw opening in the rabbit

Sarcomere lengths were measured microscopically in formalin-fixed jaw muscles of 14 rabbits divided into two groups; jaw open, and jaw closed. The measurements were compared by means of a nested analysis of variance. The sarcomeres of the jaw open group were longer in the masseter and temporalis muscles (jaw elevators) and shorter in the digastric muscle (jaw depressor) than were those of the jaw closed group. In the jaw closed position, sarcomeres in the deep portion of the masseter muscle become markedly shorter than those in the superficial part of the muscle. The values for sarcomere length in the masseter muscle of the jaw open group and the digastric of the jaw closed group are near the top of the ascending limb of isometric length-tension relation for the rabbit digastric muscle.     

Heterogeneity of fiber and sarcomere length in the human masseter muscle

This study deals with regional differences in the architectural design of the human masseter muscle. For a number of defined muscle regions the three-dimensional corrdinates of origin and insertion points, and the lengths of the muscle fibers and the sarcomeres were determined in the closed jaw position. Measurements were made from cadavers and the data were used as input for a model predicting sarcomere length at other mandibular positions. At a closed jaw average muscle fiber length of the muscle regions ranged between 19.0–30.3 mm. The fibers appeared to be considerably longer (35%) anteriorly than posteriorly in the muscle, and deeply situated fibers were on average 5% shorter than superficially situated ones. Average sarcomere length of the regions ranged between 2.27–2.55 μm, indicating that at a closed jaw position sarcomeres are at suboptimum length and have different positions on the length-tension curve. In the deep layer of the muscle sarcomeres were significantly shorter (6%) than in the superficial layer. Within the superficial layer sarcomere lengths did not differ significantly, but in the deep layer sarcomeres were shorter (8%) posteriorly than anteriorly in the muscle. The model shows that jaw displacement had a different effect on sarcomere length in the muscle regions. When the jaw was rotated about a transverse axis (open/close rotation) sarcomere excursions were relatively small in the posterior muscle regions and large in the anterior regions. The reverse was true when the jaw was rotated contralaterally about a vertical axis. It is concluded that, due to heterogeneity in fiber and sarcomere lengths, the distribution of maximal isometric tension across the muscle at full effort is not uniform.     

Activity in torso muscles during relaxed standing

Summary Electromyographic activity of erector spinae, external oblique, and rectus abdominis muscles was studied during relaxed standing compared to lying down. Activity in the forearm extensors and forearm flexors was also studied. Surface electrodes were used.Each of the torso muscles exhibited 0.2 V of activity and the forearm muscles 0.1 V while subjects were relaxed and lying down. During quiet standing the erector spinae, external oblique, and rectus abdominis muscles showed a median activity of 1.0 V, 2.5 V, and 0.7 V respectively (for a minimum of ten 10-sec samples per subject). Examination of the integrated records during standing revealed no periods without increased muscle activity in the torso muscles. By contrast, activity in the forearm muscles did not increase during standing.The major superficial muscles of posture in the torso appear to act as guy wires, being continually active during standing. There is no support for hypotheses of passive support for the torso, nor do torso muscles act in either/or fashion; both anterior and posterior muscles are active at once. There is no sign of generally increased muscle tone in all muscles or in extensors; only the postural muscles are continuously active.     

Residual force enhancement in skeletal muscles: one sarcomere after the other

The force-length relation is one of the most prominent features of striated muscles, and predicts that the force produced by a fully activated muscle is proportional to the overlap between myosin and actin filaments within sarcomeres. However, there are situations in which the force-length relation deviates from predictions based purely on filament overlap. Notably, stretch of activated skeletal muscles induces a long-lasting increase in force, which is larger than the force produced during isometric contractions at a similar length. The mechanism behind this residual force enhancement and deviations from the original force-length relation are unknown, generating heated debate in the literature. We performed a series of experiments with short segments of myofibrils and isolated sarcomeres to investigating the mechanisms of the residual force enhancement and the force length-relation. In this paper, evidence will be presented showing that force enhancement is caused by: (i) half-sarcomere non-uniformities, and (ii) a sarcomeric component, which may be associated with Ca(2+)-induced stiffness of titin molecules. These mechanisms have large implications for understanding the basic mechanisms of muscle contraction.     

Heterogeneity of fiber and sarcomere length in the human masseter muscle.

This study deals with regional differences in the architectural design of the human masseter muscle. For a number of defined muscle regions the three-dimensional coordinates of origin and insertion points, and the lengths of the muscle fibers and the sarcomeres were determined in the closed jaw position. Measurements were made from cadavers and the data were used as input for a model predicting sarcomere length at other mandibular positions. At a closed jaw average muscle fiber length of the muscle regions ranged between 19.0-30.3 mm. The fibers appeared to be considerably longer (35%) anteriorly than posteriorly in the muscle, and deeply situated fibers were on average 5% shorter than superficially situated ones. Average sarcomere length of the regions ranged between 2.27-2.55 microns, indicating that at a closed jaw position sarcomeres are at suboptimum length and have different positions on the length-tension curve. In the deep layer of the muscle sarcomeres were significantly shorter (6%) than in the superficial layer. Within the superficial layer sarcomere lengths did not differ significantly, but in the deep layer sarcomeres were shorter (8%) posteriorly than anteriorly in the muscle. The model shows that jaw displacement had a different effect on sarcomere length in the muscle regions. When the jaw was rotated about a transverse axis (open/close rotation) sarcomere excursions were relatively small in the posterior muscle regions and large in the anterior regions. The reverse was true when the jaw was rotated contralaterally about a vertical axis. It is concluded that, due to heterogeneity in fiber and sarcomere lengths, the distribution of maximal isometric tension across the muscle at full effort is not uniform.     

Micropattern width dependent sarcomere development in human ESC-derived cardiomyocytes

In this study, human embryonic stem cell-derived cardiomyocytes were seeded onto controlled two-dimensional micropatterned features, and an improvement in sarcomere formation and cell alignment was observed in specific feature geometries. High-resolution photolithography techniques and microcontact printing were utilized to produce features of various rectangular geometries, with areas ranging from 2500 μm² to 160,000 μm². The microcontact printing method was used to pattern non-adherent poly(ethylene glycol) regions on gold coated glass slides. Matrigel and fibronectin extracellular matrix (ECM) proteins were layered onto the gold-coated glass slides, providing a controlled geometry for cell adhesion. We used small molecule-based differentiation and an antibiotic purification step to produce a pure population of immature cardiomyocytes from H9 human embryonic stem cells (hESCs). We then seeded this pure population of human cardiomyocytes onto the micropatterned features of various sizes and observed how the cardiomyocytes remodeled their myofilament structure in response to the feature geometries. Immunofluorescence was used to measure α-actinin expression, and phalloidin stains were used to detect actin presence in the patterned cells. Analysis of nuclear alignment was also used to determine how cell direction was influenced by the features. The seeded cells showed clear alignment with the features, dependent on the width rather than the overall aspect ratio of the features. It was determined that features with widths between 30 μm and 80 μm promoted highly aligned cardiomyocytes with a dramatic increase in sarcomere alignment relative to the long axis of the pattern. This creation of highly-aligned cell aggregates with robust sarcomere structures holds great potential in advancing cell-based pharmacological studies, and will help researchers to understand the means by which ECM geometries can affect myofilament structure and maturation in hESC-derived cardiomyocytes.     

Ribosomes in the skeletal muscle filament lattice

The compartmentalization of myosin isoforms within a muscle cell (Gauthier: J. Cell Biol. 110:693–701, 1990) suggests that myosin might be assembled directly into thick filaments at sites where it is synthesized. We therefore examined myofibrils by immunoelectron microscopy to determine whether ribosomes are associated with thick filaments under conditions in which new myosin can be identified. We used the embryonic chick anterior latissimus dorsi (ALD), a slow muscle that is induced, by curare, to synthesize a fast myosin isoform that is not normally present. Myosin was localized in situ, using a gold-labeled monoclonal antibody that recognizes the new isoform. The gold marker, as expected, was localized preferentially to the A band. There was an overall increase of fivefold in the number of gold particles per μm² of A band in the curare-treated compared to the normal ALD, indicating that the labeled isoform was largely newly formed. There was a corresponding preferential distribution of ribosomes at the A band, especially in the H-band region, and the number of ribosomes per μm² of A band was nearly twice as high in the curare-treated as in the normal muscle. Ribosomes were located between thick filaments, often aligned in rows. We conclude that ribosomes are located within the filament lattice, and therefore that they are available for local myosin synthesis. © 1993 Wiley-Liss Inc.     

Beyond the sarcomere: CSRP3 mutations cause hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a frequent genetic cardiac disease and the most common cause of sudden cardiac death in young individuals. Most of the currently known HCM disease genes encode sarcomeric proteins. Previous studies have shown an association between CSRP3 missense mutations and either dilated cardiomyopathy (DCM) or HCM, but all these studies were unable to provide comprehensive genetic evidence for a causative role of CSRP3 mutations. We used linkage analysis and identified a CSRP3 missense mutation in a large German family affected by HCM. We confirmed CSRP3 as an HCM disease gene. Furthermore, CSRP3 missense mutations segregating with HCM were identified in four other families. We used a newly designed monoclonal antibody to show that muscle LIM protein (MLP), the protein encoded by CSRP3, is mainly a cytosolic component of cardiomyocytes and not tightly anchored to sarcomeric structures. Our functional data from both in vitro and in vivo analyses suggest that at least one of MLP's mutated forms seems to be destabilized in the heart of HCM patients harbouring a CSRP3 missense mutation. We also present evidence for mild skeletal muscle disease in affected persons. Our results support the view that HCM is not exclusively a sarcomeric disease and also suggest that impaired mechano-sensory stress signalling might be involved in the pathogenesis of HCM.