New X-ray diffraction observations on vertebrate muscle: organisation of C-protein (MyBP-C) and troponin and evidence for unknown structures in the vertebrate A-band

Previous low-angle X-ray diffraction studies of various vertebrate skeletal muscles have shown the presence of two rich layer-line patterns, one from the myosin heads and based on a 429 A axial repeat, and one from actin filaments and based on a repeat of about 360-370 A. In addition, meridional intensities have been seen from C-protein (MyBP-C; at about 440 A and its higher orders) and troponin (at about 385 A and its orders). Using preparations of intact, relaxed, bony fish fin muscles and the ID-02 low-angle X-ray camera at the ESRF with a 10 m camera length we have now seen numerous, hitherto unreported, sampled, X-ray layer-lines many of which do not fit onto the previously observed repeats and which require interpretation. The new reflections all fall on the normal ("vertical") hexagonal lattice row-lines in the highly sampled, almost "crystalline", low-angle diffraction X-ray patterns from bony fish muscle, indicating that they all arise from the muscle A-band. However, they do not fall on a single axial repeat. In direct confirmation of our previous analysis, some of these new reflections are explained by the interaction in resting muscle between the N-terminal ends of myosin-bound C-protein molecules with adjacent actin filaments, possibly through the Pro-Ala-rich region. Other newly observed reflections lie on a much longer repeat, but they are most easily interpreted in terms of the arrangement of troponin on the actin filaments. If this is so, then the implication is that the actin filaments and their troponin complexes are systematically arranged in the fish muscle A-band lattice relative to the myosin head positions, and that these newly observed X-ray reflections, when fully analysed, will report on the shape and distribution of troponin molecules in the resting muscle A-band. The less certain contributions of titin and nebulin to these new reflections have also been tested and are described. Many of the new reflections do not appear to come from these known structures. There must be structural features of the A-band that have not yet been described.     

A structural origin of latency relaxation in frog skeletal muscle

A time-resolved x-ray diffraction study at a time resolution of 0.53 ms was made to investigate the structural origin of latency relaxation (LR) in frog skeletal muscle. Intensity and spacing measurements were made on meridional reflections from the Ca-binding protein troponin and the thick filament and on layer lines from the thin filament. At 16 degrees C, the intensity and spacing of all reflections started to change at 4 ms, simultaneously with the LR. At 0 degrees C, the intensity of the troponin reflection and the layer lines from the thin filament and the spacing of the 14.3-nm myosin meridional reflection, but not the spacing of other myosin meridional reflections, began to change at approximately 15 ms, when the LR also started. Intensity of myosin-based reflections started to change later. When the muscle was stretched to non-overlap length, the intensity and spacing changes of the myosin reflections disappeared. The simultaneous spacing change of the 14.3-nm myosin meridional reflection with the LR suggests that detachment of myosin heads that are bound to actin in the resting muscle is the cause of the LR.     

X-ray diffraction patterns from mammalian heart muscle

We have obtained light and X-ray diffraction patterns from trabecular and papillary muscles of various mammalian hearts in the living resting state and in rigor. Equatorial X-ray diffraction patterns from living muscles show the 1,0 and 1,1 reflections from a hexagonal lattice of filaments. The lattice spacing varies with sarcomere length over the observable range (2·0 to 2·5 μm) in such a manner that the lattice volume remains constant. In the living resting state the 1,0 reflection is stronger than the 1,1 reflection, whereas in rigor the 1,1 reflection is almost as strong as the 1,0 reflection. These intensity changes are similar to those found in vertebrate skeletal muscle, suggesting that the mechanism of cross-bridge attachment to actin is similar in both muscles.Two types of meridional X-ray diffraction pattern were observed in muscles in different conditions. One type, obtained from dead or glycerol-extracted muscles or from muscles treated with iodoacetate, showed a strong actin-related pattern but only a weak pattern associated with myosin. This type of pattern was similar to that from vertebrate skeletal muscle in rigor. The other type, obtained from living, resting muscle, showed a weaker actin pattern but a stronger myosin pattern. The myosin pattern included layer-line reflections associated with projections from the thick filaments. This second type of pattern was similar to that from resting vertebrate skeletal muscle, but the layer lines were weaker. The weakness of the myosin layer lines may indicate that part of the high resting tension found in heart muscle arises from a small amount of actin-myosin interaction in the resting state. Such interaction could provide a mechanism for varying the diastolic length of heart muscle and thereby the diastolic volume of the heart.     

Immunochemical analysis of porcine cardiac C-protein

C-protein has been isolated from pig heart and its immunochemical properties studied. It is extracted with myosin, and separated from the myosin on a DEAE-Sephadex column. The amount of C-protein recovered from crude myosin is approx. 3.5%. The molecular weight of C-protein is 150,000. Anti-C-protein serum reacts with crude myosin and purified C-protein but not with purified myosin in immunodiffusion plates. Cardiac C-protein does not react with anti-skeletal white muscle C-protein serum. Immunoblotting experiments show that anti-cardiac C-protein serum reacts with a Mr = 150,000 component in myofibrils or crude myosin. C-protein is located in the A-band, except the M-line region, of the myofibrils. These results indicate that C-protein is an intrinsic component of the thick filaments in pig heart myofibrils.     

The binding of skeletal muscle C-protein to F-actin, and its relation to the interaction of actin with myosin subfragment-1.

C-protein is a component of thick filaments of skeletal muscle myofibrils. It is bound to the assembly of myosin tails that forms the filament backbone. We report here that C-protein can also bind to F-actin, with a limiting stoichiometry of approximately one C-protein molecule per 3 to 5 actin subunits and a dissociation constant in the micromolar range at ionic strength 0·07. The binding is not significantly affected by ATP, calcium ions or temperature, or by the presence of tropomyosin on the actin, but it is weakened by increasing ionic strength. Myosin subfragment-1 (S-1) competes with C-protein for binding to actin. In the absence of ATP, S-1 displaces nearly all bound C-protein from actin, while in the presence of ATP, C-protein inhibits the actin activation of S-1 ATPase. Although there is no direct evidence that interaction of C-protein with actin is physiologically significant, the lenght of the C-protein molecule is sufficient so that it could make contact with the thin filaments in muscle while remaining attached to the thick filaments.     

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.     

Frog skeletal muscle thick filaments are three-stranded

A procedure has been developed for isolating and negatively staining vertebrate skeletal muscle thick filaments that preserves the arrangement of the myosin crossbridges. Electron micrographs of these filaments showed a clear periodicity associated with crossbridges with an axial repeat of 42.9 nm. Optical diffraction patterns of these images showed clear layer lines and were qualitatively similar to published x-ray diffraction patterns, except that the 1/14.3-nm meridional reflection was somewhat weaker. Computer image analysis of negatively stained images of these filaments has enabled the number of strands to be established unequivocally. Both reconstructed images from layer line data and analysis of the phases of the inner maxima of the first layer line are consistent only with a three-stranded structure and cannot be reconciled with either two- or four-stranded models.     

Interaction of aldolase with thin filaments within I-disks, isolated from skeletal muscles

Using electron microscopy and optical diffraction, Ca2+-dependent binding of a glycolytic enzyme (aldolase) to thin filaments of isolated skeletal muscle I-disks have been revealed. On the micrographs of negatively stained I-disks the cross-striation determined by troponin-tropomyosin complex distribution has a period of about 38 nm. The width of troponin-tropomyosin stripes is 5-6 nm. On the optical diffraction patterns from isolated I-disks the meridional reflections measuring 38.5, 19.2, 12.8 nm are present. On the micrographs of isolated I-disks, treated with aldolase in the absence of Ca2+ (1 mM EGTA) the width of periodic transverse stripes (period approximately 38 nm) increases from 5-6 nm to 25-28 nm due to the interaction of aldolase with thin filaments. On the optical diffraction patterns from I-disks treated with aldolase in the absence of Ca2+ (1 mM EGTA) the strong meridional reflection equal to 38.5 nm is present, while the reflections equal to 19.2 nm are absent. The optical diffraction patterns from I-disks treated with aldolase in the presence of Ca2+ (greater than or equal to 10(-5) M) do not, as a rule, differ from those obtained from I-disks not treated with aldolase, i.e. they contain the three above reflections. The binding of aldolase to thin filaments in the absence of Ca2+ is the reason of disappearance of meridional reflections equal to 19.2 and 12.8 nm.     

Changes of thick filament structure during contraction of frog striated muscle.

The strongest myosin-related features in the low-angle axial x-ray diffraction pattern of resting frog sartorius muscle are the meridional reflections corresponding to axial spacings of 21.4 and 14.3 nm, and the first layer line, at a spacing 42.9 nm. During tetanus the intensities of the first layer line and the 21.4-nm meridional decrease by 62 and 80% respectively, but, when the muscle is fresh, the 14.3-nm meridional intensity rises by 13%, although it shows a decrease when the muscle is fatigued. The large change in the intensity of the 21.4-nm meridional reflection suggests that the projected myosin cross-bridge density onto the thick filament axis changes during contraction. The model proposed by Bennett (Ph.D. Thesis, University of London, 1977) in which successive cross-bridge levels are at 0,3/8, and 5/8 of the 42.9-nm axial repeat in the resting muscle, passing to 0, 1/3, and 2/3 in the contracting state, can explain why the 21.4-nm reflection decreases in intensity while the 14.3-nm increases when the muscle is activated. The model predicts a rather larger increase of the 14.3-nm reflection intensity during contraction than that observed, but the discrepancy may be removed if a small change of shape or tilt of the cross-bridges relative to the thick filament axis is introduced. The decrease of the intensity of the first layer line indicates that the cross-bridges become disordered in the plane perpendicular to the filament axis.     

X-ray study of the effect of pyrophosphate on thick filament structure in skinned fiber bundles of the rabbit psoas muscle

Structure of thick filaments in the chemically skinned fibre bundles of rabbit psoas muscle in a state of pseudorelaxation induced by adding 2 mM pyrophosphate (PP) and of PP-mixture with 40% ethyleneglycol to the bathing rigor solution was studied with the help of X-ray diffraction technique. Reduction in the isometric rigor tension by about 50-70% in a state of pseudorelaxation is accompanied by significant changes in the relative intensities of a number of meridional reflections, indicating that in situ the structure and location of S-2 segment may be regulated by the structural changes in the acto S-1-complex during its cyclic interaction with ATP.     

Diffraction rings obtained from a suspension of skeletal myofibrils by laser light illumination. Study of internal structure of sarcomeres.

Diffraction rings corresponding to the first, second, and third order were obtained by laser light illumination from a suspension of rabbit glycerinated psoas myofibrils (diameter, 1-2 microns; average length of the straight region, 44 microns; average sarcomere length, 2.2-2.6 microns) of which the optical thickness was appropriately chosen. Dispersed myofibrils were nearly randomly oriented in two dimensions, so that the effects of muscle volume were minimized; these effects usually interfere significantly with a quantitative analysis of laser optical diffraction in the fiber system. The diameters of diffraction rings represented the average sarcomere length. By using this system, we confirmed the ability of the unit cell (sarcomere) structure model to explain the intensity change of diffraction lines accompanying the dissociation from both ends of thick filaments in a high salt solution. The length of an A-band estimated from the relative intensity of diffraction rings and that directly measured on phase-contrast micrographs coincided well with each other. Also, we found that myofibrils with a long sarcomere length shorten to a slack length accompanying the decrease in overlap between thick and thin filaments produced by the dissociation of thick filaments.     

Immunohistochemical analysis of C-protein isoforms in cardiac and skeletal muscle of the axolotl,Ambystoma mexicanum

Of the several proteins located within sarcomeric A-bands, C-protein, a myosin binding protein (MyBP) is thought to regulate and stabilize thick filaments during assembly. This paper reports the characterization of C-protein isoforms in juvenile and adult axolotls, Ambystoma mexicanum, by means of immunofluorescent microscopy and Western blot analyses. C-protein and myosin are found specifically within the A-bands, whereas tropomyosin and -actin are detected in the I-bands of axolotl myofibrils. The MF1 antibody prepared against the fast skeletal muscle isoform of chicken C-protein specifically recognizes a cardiac isoform (Axcard1) in juvenile and adult axolotls but does not label axolotl skeletal muscle. The ALD66 antibody, which reacts with the C-protein slow isoform in chicken, localizes only in skeletal muscle of the axolotl. This slow axolotl isoform (Ax_slow) displays a heterogeneous distribution in fibers of dorsalis trunci skeletal muscle. The C₃₁₅ antibody against the chicken C-protein cardiac isoform identifies a second axolotl cardiac isoform (Ax_card2), which is present also in axolotl skeletal muscle. No C-protein was detected in smooth muscle of the juvenile and adult axolotl with these antibodies.This work was supported by NIH grants HL-32184 and HL-37702 and a grant-in-aid from the American Heart Association to L.F.L.     

The mammalian cardiac muscle thick filament: crossbridge arrangement

Although skeletal muscle thick filaments have been extensively studied, information on the structure of cardiac thick filaments is limited. Since cardiac muscle differs in many physiological properties from skeletal muscle it is important to elucidate the structure of the cardiac thick filament. The structure of isolated and negatively stained rabbit cardiac thick filaments has been analyzed from computed Fourier transforms and image analysis. The transforms are detailed, showing a strong set of layer lines corresponding to a 42.9 nm quasi-helical repeat. The presence of relatively strong "forbidden" meridional reflections not expected from ideal helical symmetry on the second, fourth, fifth, seventh, eighth, and tenth layer lines suggest that the crossbridge array is perturbed from ideal helical symmetry. Analysis of the phase differences for the primary reflections on the first layer line of transforms from 15 filaments showed an average difference of 170 degrees, close to the value of 180 degrees expected for an odd-stranded structure. Computer-filtered images of the isolated thick filaments unequivocally demonstrate a three-stranded arrangement of the crossbridges on the filaments and provide evidence that the crossbridge arrangement is axially perturbed from ideal helical symmetry.     

A new protein of the thick filaments of vertebrate skeletal myofibrils. Extractions, purification and characterization

A new protein component of skeletal myofibrils has been isolated and characterized. It is prepared from impure myosin preparations and corresponds to band C, the principal contaminant observed in sodium dodecyl sulphate polyacrylamide gel electrophoresis patterns of such preparations (Starr and Offer, 1971).The C-protein, as we term it, is deduced to be a component of the skeletal myofibril because (i) glycerinated or fresh myoflbrils contain a component with a mobility identical to C-protein on sodium dodecyl sulphate gels, (ii) this component is extracted from myofibrils by the same solvent which extracts C-protein and (iii) C-protein may be prepared from preparations of isolated myofibrils. It is presumed to be a component of the thick filaments because it binds strongly to myosin at low ionic strength; immunological evidence which confirms this view is presented elsewhere.The quantity of C-protein in the myofibril has been estimated to be 2.0% by densitometry of sodium dodecyl sulphate gels of glycerinated myofibrils using actin as an internal reference. About forty molecules of C-protein are present in a thick filament.The properties of C-protein distinguish it from the other well-characterized myoflbrillar proteins. The C-protein molecule contains a single polypeptide chain of molecular weight 140,000. The intrinsic viscosity of 13.6 ml/g suggests that the molecule is neither completely globular nor as elongated as molecules like paramyosin or tropomyosin. The α-helical content is very low and the proline content higher than the other myofibrillar proteins. The molecule associates at low ionic strength.C-protein has no ATPase activity, nor does it affect the ATPase of pure myosin. But it reduces the activity of the actin-activated myosin ATPase by about half, this inhibition being independent of the level of Ca²⁺. C-protein does not bind Ca²⁺ in the presence of Mg²⁺. Its possible location and function are discussed.     

Structure of actin paracrystals induced by nerve growth factor

When nerve growth factor is added to F-actin, well-ordered bundles of filaments are formed. These bundles are observed even at low concentrations of NGF²¹, but when N-bromosuccinimide-treated NGF, a biologically inactive form of the protein is used, a much higher concentration is required to produce aggregation. Moreover, the bundles induced by the modified NGF are not very well ordered and show amorphous aggregates attached at various points.Electron microscopy of paracrystals induced by native NGF shows that, although they resemble pure actin paracrystals induced by Mg²⁺, the interfilament spacing is larger and bridges connect the filaments. Optical diffraction patterns show, in addition to the off-meridional reflections characteristic of the actin helix, meridional reflections on the first and fourth layer-lines, at axial spacings of 37 and 9 nm. Measurements of the axial positions of the layer-lines show that the actin helical symmetry is not significantly different from that in pure actin paracrystals. The presence of the meridional reflections indicates that groups of two or three bridges with spacing 9 nm or nearly 9 nm are arranged along the bundles at a repeating interval of 37 nm.Actin filament bundles have been observed in several non-muscle cells, and specific actin-binding proteins have been identified as responsible for this aggregation. Our in vitro observations show that the biologically active form of NGF interacts with actin and organizes it into well-ordered paracrystalline arrays. The in vitro formation of NGF-actin complexes may be related to the in vivo mechanism of action of this growth factor.     

Characterization of a non-indexible equatorial x-ray reflection from frog sartorius muscle.

X-ray equatorial reflections from frog sartorius muscle were studied using a position sensitive detector. A weak reflection appeared between the 10 and 11 peaks which did not index on the hexagonal filament lattice. This reflection, first reported by Elliott et al. (1967), was further characterized. The spacing of the reflection varied in direct proportion to that of the 10 peaks for sarcomere lengths between 2·0 μm and 3·0 μm. Its intensity appeared relatively insensitive to length changes. Optical diffraction patterns from electron micrographs of oblique sections through muscle gave ratios for the spacings of the myosin filaments and the Z-disc lattice that correlated very closely with the X-ray results. It is suggested that the Z-disc structure is the major source of this nonindexible reflection.     

Role of desmin filaments in chicken cardiac myofibrillogenesis

Desmin filaments are muscle-specific intermediate filaments located at the periphery of the Z-discs, and they have been postulated to play a critical role in the lateral registration of myofibrils. Previous studies suggest that intermediate filaments may be involved in titin assembly during the early stages of myofibrillogenesis. In order to investigate the putative function of desmin filaments in myofibrillogenesis, rabbit anti-desmin antibodies were introduced into cultured cardiomyocytes by electroporation to perturb the normal function of desmin filaments. Changes in the assembly of several sarcomeric proteins were examined by immunofluorescence. In cardiomyocytes incorporated with normal rabbit serum, staining for alpha-actinin and muscle actin displayed the typical Z-line and I-band patterns, respectively, while staining for titin with monoclonal anti-titin A12 antibody, which labels a titin epitope at the A-I junction, showed the periodic doublet staining pattern. Staining for C-protein gave an amorphous pattern in early cultures and identified A-band doublets in older cultures. In contrast, in cardiomyocytes incorporated with anti-desmin antibodies, alpha-actinin was found in disoriented Z-discs and the myofibrils became fragmented, forming mini-sarcomeres. In addition, titin was not organized into the typical A-band doublet, but appeared to be aggregated. Muscle actin staining was especially weak and appeared in tiny clusters. Moreover, in all ages of cardiomyocytes tested, C-protein remained in the disassembled form. The present data suggest the essential role of desmin in myofibril assembly.     

Structural changes of the regulatory proteins bound to the thin filaments in skeletal muscle contraction by X-ray fiber diffraction

In order to clarify the structural changes related to the regulation mechanism in skeletal muscle contraction, the intensity changes of thin filament-based reflections were investigated by X-ray fiber diffraction. The time course and extent of intensity changes of the first to third order troponin (TN)-associated meridional reflections with a basic repeat of 38.4 nm were different for each of these reflections. The intensity of the first and second thin filament layer lines changed in a reciprocal manner both during initial activation and during the force generation process. The axial spacings of the TN-meridional reflections decreased by ∼0.1% upon activation relative to the relaxing state and increased by ∼0.24% in the force generation state, in line with that of the 2.7-nm reflection. Ca²⁺-binding to TN triggered the shortening and a change in the helical symmetry of the thin filaments. Modeling of the structural changes using the intensities of the thin filament-based reflections suggested that the conformation of the globular core domain of TN altered upon activation, undergoing additional conformational changes at the tension plateau. The tail domain of TN moved together with tropomyosin during contraction. The results indicate that the structural changes of regulatory proteins bound to the actin filaments occur in two steps, the first in response to the Ca²⁺-binding and the second induced by actomyosin interaction.     

Mammalian cardiac muscle thick filaments: their periodicity and interactions with actin

Cardiac muscle has been extensively studied, but little information is available on the detailed macromolecular structure of its thick filament. To elucidate the structure of these filaments I have developed a procedure to isolate the cardiac thick filaments for study by electron microscopy and computer image analysis. This procedure uses chemical skinning with Triton X-100 to avoid contraction of the muscle that occurs using the procedures previously developed for isolation of skeletal muscle thick filaments. The negatively stained isolated filaments appear highly periodic, with a helical repeat every third cross-bridge level (43 nm). Computed Fourier transforms of the filaments show a strong set of layer lines corresponding to a 43-nm near-helical repeat out to the 6th layer line. Additional meridional reflections extend to at least the 12th layer line in averaged transforms of the filaments. The highly periodic structure of the filaments clearly suggests that the weakness of the layer lines in x-ray diffraction patterns of heart muscle is not due to an inherently more disordered cross-bridge arrangement. In addition, the isolated thick filaments are unusual in their strong tendency to remain bound to actin by anti-rigor oriented cross-bridges (state II or state III cross-bridges) under relaxing conditions.     

Isolation and composition of thick filaments from rabbit skeletal muscle

A method has been developed for the isolation of thick filaments from rabbit skeletal muscle. We found that the thick filaments of this muscle are readily dispersed in the presence of a relaxing medium if the M and Z-line structures are first extracted in a low-salt solvent system. Thick filaments were separated from thin filaments by zone sedimentation in a 10% to 30% glycerol density gradient. The isolated filaments are homogeneous in length (1.5 to 1.6 μm) and retain the physical characteristics of these structures observed in sectioned muscle. Gel electrophoresis of thick filaments in the presence of sodium dodecyl sulfate showed a band of C-protein as well as bands with mobilities characteristic of the heavy and light chains of myosin. No other protein species was detected in these experiments. Thus our results provide evidence against the presence of a special protein component which would serve as the core of the skeletal thick filament structure. From the relative stain density of bands, the molar ratio of C-protein to myosin was estimated to be 1 to 5.8.