Myosin thick filaments from adult rabbit skeletal muscles

Myosin subfragment 1 (S1) forms dimers in the presence of Mg(2+) or MgADP or MgATP. The entire myosin molecule forms head-head dimers in the presence of MgATP. The angle between the two subunits in the S1 dimer is 95 degrees. Assuming that the length of the globular part of S1 is approximately 12 nm and that the S1/S2 joint (lever arm approximately 7 nm) is clearly bent, the cylinder tangent to this dimer should have a diameter of approximately 18 nm, close to the approximately 16-20 nm suggested by many studies for the diameter of thick filaments in situ. These conclusions led us to re-examine our previous model, according to which two heads from two opposite myosin molecules are inserted into the filament core and interact as dimers. We studied synthetic filaments by electron microscopy, enzyme activity assays, controlled digestion and filament-filament interaction analysis. Synthetic filaments formed by rapid dilution in the presence of 1 mM EDTA at room temperature ( approximately 22 degrees C) had all their myosin heads outside the backbone. These filaments are called superfilaments (SF). Synthetic filaments formed by slow dilution, in the presence of either 2 mM Mg(2+) or 0.5 mM MgATP and at low temperature ( approximately 0 degrees C) had one myosin head outside the backbone and one head inside. These filaments are called filaments (F). Synthetic filaments formed by slow dilution, in the presence of 4 mM MgATP at low temperature ( approximately 0 degrees C) had most of their heads inserted in the filament core. These filaments are called antifilaments (AF). These experimental results provide important new information about myosin synthetic filaments. In particular, we found that myosin heads were involved in filament assembly and that filament-filament interactions can occur via the external heads. Native filaments (NF) from rabbit psoas muscle were also studied by enzyme assays. Their structure depended on the age of the rabbit. NF from 4-month-old rabbits were three-stranded, i.e. six myosin heads per crown, two of which were inside the core and four outside. NF from 18-month-old rabbits were two-stranded (similar to F).     

Reduced cross-bridge dependent stiffness of skinned myocardium from mice lacking cardiac myosin binding protein-C

The role of cardiac myosin binding protein-C (MyBP-C) on myocardial stiffness was examined in skinned papillary muscles of wild-type (WT(+/+)) and homozygous truncated cardiac MyBP-C (MyBP-C(t/t) male mice. No MyBP-C was detected by gel electrophoresis or by Western blots in the MyBP-C(t/t) myocardium. Rigor-bridge dependent myofilament stiffness, i.e., rigor minus relaxed stiffness, in the MyBP-C(t/t) myocardium (281 +/- 44 kN/m2) was 44% that in WT(+/+) (633 +/- 141 kN/m2). The center-to-center spacing between thick filaments as determined by X-ray diffraction in MyBP-C(t/t) (45.0 +/- 1.2 nm) was not significantly different from that in WT(+/+) (43.2 +/- 0.9 nm). The fraction of cross-sectional area comprised of myofibrils, as determined by electron microscopy, was reduced in the MyBP-C(t/t) (39.9%) by 10% compared to WT(+/+) (44.5%). These data suggest that the 56% reduction in rigor-bridge dependent stiffness of the skinned MyBP-C(t/t) myocardium could not be due solely to a 10% reduction in the number of thick filaments per cross-sectional area and must also be due to approximately 50% reduction in the stiffness of the rigor-bridge attached thick filaments lacking MyBP-C.     

Reduced cross-bridge dependent stiffness of skinned myocardium from mice lacking cardiac myosin binding protein-C

The role of cardiac myosin binding protein-C (MyBP-C) on myocardial stiffness was examined in skinned papillary muscles of wild-type (WT^+/+) and homozygous truncated cardiac MyBP-C (MyBP-C^t/t) male mice. No MyBP-C was detected by gel electrophoresis or by Western blots in the MyBP-C^t/t myocardium. Rigor-bridge dependent myofilament stiffness, i.e., rigor minus relaxed stiffness, in the MyBP-C^t/t myocardium (281 ± 44 kN/m²) was 44% that in WT^+/+ (633 ± 141 kN/m²). The center-to-center spacing between thick filaments as determined by X-ray diffraction in MyBP-C^t/t (45.0 ± 1.2 nm) was not significantly different from that in WT^+/+ (43.2 ± 0.9 nm). The fraction of cross-sectional area comprised of myofibrils, as determined by electron microscopy, was reduced in the MyBP-C^t/t (39.9%) by 10% compared to WT^+/+ (44.5%). These data suggest that the 56% reduction in rigor-bridge dependent stiffness of the skinned MyBP-C^t/t myocardium could not be due solely to a 10% reduction in the number of thick filaments per cross-sectional area and must also be due to approximately 50% reduction in the stiffness of the rigor-bridge attached thick filaments lacking MyBP-C. (Mol Cell Biochem 263: 73–80, 2004)     

Myosin light chain kinase binding to actin filaments

Smooth muscle myosin light chain kinase (MLCK) plays important roles in contractile-motile processes of a variety of cells. Three DFRxxL motifs at the kinase N-terminus (residues 2–63) are critical for high-affinity binding to actin-containing filaments [Smith et al. (1999) J. Biol. Chem. 274, 29433–29438]. A GST fusion protein containing residues 1–75 of MLCK (GST75-MLCK) bound maximally to both smooth muscle myofilaments and F-actin at 0.28 and 0.31 mol GST75-MLCK/mol actin with respective K_D values of 0.1 μM and 0.8 μM. High-affinity binding of MLCK to actin-containing filaments may be due to each DFRxxL motif binding to one actin monomer in filaments.     

Direction and speed of actin filaments moving along thick filaments isolated from molluscan smooth muscle

The active movement of fluorescence-labeled actin filaments along thick filaments isolated from molluscan smooth muscle was observed. Along a single thick filament, actin filaments moved toward the center of the thick filament at the speed of 1.19 +/- 0.38 microns s-1 (mean +/- SD, n = 42) and detached themselves from it upon reaching the central zone. Movement of actin also occurred in the opposite direction, i.e., away from the center, albeit at a much lower velocity (0.09 +/- 0.07 microns s-1, n = 17). Thus, the thick filament shows functional bipolarity in terms of velocity but does not determine the direction of the movement.     

Myosin S2 is not required for effects of myosin binding protein-C on motility

The unique myosin binding protein-c "motif" near the N-terminus of myosin binding protein-C (MyBP-C) binds myosin S2. Previous studies demonstrated that recombinant proteins containing the motif and flanking regions (e.g., C1C2) affect thin filament movement in motility assays using heavy meromyosin (S1 plus S2) as the molecular motor. To determine if S2 is required for these effects we investigated whether C1C2 affects motility in assays using only myosin S1 as the motor protein. Results demonstrate that effects of C1C2 are comparable in both systems and suggest that the MyBP-C motif affects motility through direct interactions with actin and/or myosin S1.     

Crystal structure of the C1 domain of cardiac myosin binding protein-C: implications for hypertrophic cardiomyopathy

C-protein is a major component of skeletal and cardiac muscle thick filaments. Mutations in the gene encoding cardiac C-protein [cardiac myosin binding protein-C (cMyBP-C)] are one of the principal causes of hypertrophic cardiomyopathy. cMyBP-C is a string of globular domains including eight immunoglobulin-like and three fibronectin-like domains termed C0-C10. It binds to myosin and titin, and probably to actin, and may have both a structural and a regulatory role in muscle function. To help to understand the pathology of the known mutations, we have solved the structure of the immunoglobulin-like C1 domain of MyBP-C by X-ray crystallography to a resolution of 1.55 Å. Mutations associated with hypertrophic cardiomyopathy are clustered at one end towards the C-terminus, close to the important C1C2 linker, where they alter the structural integrity of this region and its interactions.     

Myosin and paramyosin of Caenorhabditis elegans embryos assemble into nascent structures distinct from thick filaments and multi-filament assemblages

The organization of myosin heavy chains (mhc) A and B and paramyosin (pm) which are the major proteins of thick filaments in adult wild-type Caenorhabditis elegans were studied during embryonic development. As a probe of myosin-paramyosin interaction, the unc-15 mutation e73 which produces a glu342lys charge change in pm and leads to the formation of large paracrystalline multi-filament assemblages was compared to wild type. These three proteins colocalized in wild-type embryos from 300 to 550 min of development after first cleavage at 20 degrees C on the basis of immunofluorescence microscopy using specific monoclonal antibodies. Linear structures which were diversely oriented around the muscle cell peripheries appeared at 360 min and became progressively more aligned parallel to the embryonic long axis until distinct myofibrils were formed at 550 min. In the mutant, mhc A and pm were colocalized in the linear structures, but became progressively separated until they showed no spatial overlap at the myofibril stage. These results indicate that the linear structures represent nascent assemblies containing myosin and pm in which the proteins interact differently than in wild-type thick filaments of myofibrils. In e73, these nascent structures were distinct from the multi-filament assemblages. The overlapping of actin and mhc A in the nascent linear structures suggests their possible structural and functional relationship to the "stress fiber-like structures" of cultured vertebrate muscle cells.     

The structure of thick filaments on longitudinal sections of rabbit psoas muscle

By means of electron microscopy the longitudinal sections of chemically skinned fibres of rigorised rabbit psoas muscle have been examined at pH of rigorising solutions equal to 6, 7, 8 (I = 0.125) and ionic strengths equal to 0.04, 0.125, 0.34 (pH 7.0). It has been revealed that at pH 6.0 the bands of minor proteins localization in A-disks were seen very distinctly, while at pH 7.0 and I = 0.125 these bands can be revealed only by means of antibody labelling technique. At the ionic strength of 0.34 (pH 7.0) the periodicity of 14.3 nm in thick filaments was clearly observed, which was determined by packing of the myosin rods into the filament shaft and of the myosin heads (cross-bridges) on the filament surface. The number of cross-bridge rows in the filament equals 102. A new scheme of myosin cross-bridge distribution in thick filaments of rabbit psoas muscle has been suggested according to which two rows of cross-bridges at each end of a thick filament are absent. The filament length equals 1.64 +/- 0.01 micron. It has been shown that the length of thick filament as well as the structural organization of their end regions in rabbit psoas muscle and frog sartorius one are different.     

Assembly and structure of calcium-induced thick vimentin filaments.

Using a viscometric assay and various electron microscopic procedures (negative staining, rotary shadowing, ultrathin sectioning) we have determined the influences of different kinds of ions and of ionic strength on the structures formed by assembly of soluble subunits of vimentin from bovine lens tissue or from Escherichia coli transformed with Xenopus vimentin cDNA. In contrast to the assembly of typical, i.e., 8 to 14-nm, intermediate-sized filaments (IFs) at elevated (e.g., 160 mM) concentrations of monovalent cations and at millimolar Mg2+ concentrations, filaments formed in the presence of Ca2+ ions (e.g., 5 mM) appeared at a lower rate, attained lower viscosity and were considerably thicker and shorter. The largest diameter measured was that for the recombinant amphibian protein: 24.2 +/- 8.5 nm in negative staining, 28.7 +/- 5.6 nm in sections. These thick Ca(2+)-induced filaments, however, revealed the same approximately 2 nm protofilament composition and approximately 20 nm cross-striation pattern as typical IFs, indicative of a similar molecular arrangement. The significance of this unusual structural IF protein assembly is discussed.     

Mechanical unfolding of cardiac myosin binding protein-C by atomic force microscopy

Cardiac myosin-binding protein-C (cMyBP-C) is a thick-filament-associated protein that performs regulatory and structural roles within cardiac sarcomeres. It is a member of the immunoglobulin (Ig) superfamily of proteins consisting of eight Ig- and three fibronectin (FNIII)-like domains, along with a unique regulatory sequence referred to as the M-domain, whose structure is unknown. Domains near the C-terminus of cMyBP-C bind tightly to myosin and mediate the association of cMyBP-C with thick (myosin-containing) filaments, whereas N-terminal domains, including the regulatory M-domain, bind reversibly to myosin S2 and/or actin. The ability of MyBP-C to bind to both myosin and actin raises the possibility that cMyBP-C cross-links myosin molecules within the thick filament and/or cross-links myosin and thin (actin-containing) filaments together. In either scenario, cMyBP-C could be under mechanical strain. However, the physical properties of cMyBP-C and its behavior under load are completely unknown. Here, we investigated the mechanical properties of recombinant baculovirus-expressed cMyBP-C using atomic force microscopy to assess the stability of individual cMyBP-C molecules in response to stretch. Force-extension curves showed the presence of long extensible segment(s) that became stretched before the unfolding of individual Ig and FNIII domains, which were evident as sawtooth peaks in force spectra. The forces required to unfold the Ig/FNIII domains at a stretch rate of 500 nm/s increased monotonically from ∼30 to ∼150 pN, suggesting a mechanical hierarchy among the different Ig/FNIII domains. Additional experiments using smaller recombinant proteins showed that the regulatory M-domain lacks significant secondary or tertiary structure and is likely an intrinsically disordered region of cMyBP-C. Together, these data indicate that cMyBP-C exhibits complex mechanical behavior under load and contains multiple domains with distinct mechanical properties.     

The active cross-bridge motions of isolated thick filaments from myosin-regulated muscles detected by quasi-elastic light scattering.

Intensity fluctuation spectroscopy has been used successfully as a probe that can detect an increase in high-frequency internal motions of isolated thick filaments of Limulus muscle upon the addition of calcium ions. We have attributed such motions to cross-bridge motion instead of to an increase in the flexibility of the filament backbone. Here we show that after cleavage of the S-1 and then the S-2 moieties with papain, cross-linking the myosin heads to the filament backbone, or heat denaturation (42 degrees C, 10 min), the increase in the high frequency internal motions in the thick filaments no longer occurs. Congo Red, which has been shown to induce shortening of isolated myofibrils, also increases the high-frequency motions of the isolated filaments. Furthermore, the increase is suppressed by treating the filaments with a myosin ATPase inhibitor such as vanadate ions (10 mM) or by replacing ATP with either an equimolar CrADP or the nonhydrolyzable ATP analogue beta, gamma-imido-adenine-5'-triphosphate (AMP-PNP). Calcium ions have a similar effect on isolated thick filaments from scallop muscle, where the myosin is known to be regulatory. Calcium ions have no such effect on thick filaments isolated from frog muscle, which is believed not to be regulated by calcium binding to myosin. These results confirm our earlier supposition that the additional high frequency internal motions of the thick filaments isolated from striated muscle of Limulus are related to the energy dependent, active cross-bridge motions.     

Myosin molecule packing within the vertebrate skeletal muscle thick filaments. A complete bipolar model

Computer modelling related to the real dimensions of both the whole filament and the myosin molecule subfragments has revealed two alternative modes for myosin molecule packing which lead to the head disposition similar to that observed by EM on the surface of the cross-bridge zone of the relaxed vertebrate skeletal muscle thick filaments. One of the modes has been known for three decades and is usually incorporated into the so-called three-stranded model. The new mode differs from the former one in two aspects: (1) myosin heads are grouped into asymmetrical cross-bridge crowns instead of symmetrical ones; (2) not the whole myosin tail, but only a 43-nm C-terminus of each of them is straightened and near-parallel to the filament axis, the rest of the tail is twisted. Concurrent exploration of these alternative modes has revealed their influence on the filament features. The parameter values for the filament models as well as for the building units depicting the myosin molecule subfragments are verified by experimental data found in the literature. On the basis of the new mode for myosin molecule packing a complete bipolar structure of the thick filament is created.     

Elastic properties of isolated thick filaments measured by nanofabricated cantilevers.

Using newly developed nanofabricated cantilever force transducers, we have measured the mechanical properties of isolated thick filaments from the anterior byssus retractor muscle of the blue mussel Mytilus edulis and the telson levator muscle of the horseshoe crab Limulus polyphemus. The single thick filament specimen was suspended between the tip of a flexible cantilever and the tip of a stiff reference beam. Axial stress was placed on the filament, which bent the flexible cantilever. Cantilever tips were microscopically imaged onto a photodiode array to extract tip positions, which could be converted into force by using the cantilever stiffness value. Length changes up to 23% initial length (Mytilus) and 66% initial length (Limulus) were fully reversible and took place within the physiological force range. When stretch exceeded two to three times initial length (Mytilus) or five to six times initial length (Limulus), at forces approximately 18 nN and approximately 7 nN, respectively, the filaments broke. Appreciable and reversible strain within the physiological force range implies that thick-filament length changes could play a significant physiological role, at least in invertebrate muscles.     

Cardiac myosin binding protein-C: redefining its structure and function

Mutations of cardiac myosin binding protein-C (cMyBP-C) are inherited by an estimated 60 million people worldwide, and the protein is the target of several kinases. Recent evidence further suggests that cMyBP-C mutations alter Ca²⁺ transients, leading to electrophysiological dysfunction. Thus, while the importance of studying this cardiac sarcomere protein is clear, preliminary data in the literature have raised many questions. Therefore, in this article, we propose to review the structure and function of cMyBP-C with particular respect to the role(s) in cardiac contractility and whether its release into the circulatory system is a potential biomarker of myocardial infarction. We also discuss future directions and experimental designs that may lead to expanding the role(s) of cMyBP-C in the heart. In conclusion, we suggest that cMyBP-C is a regulatory protein that could offer a broad clinical utility in maintaining normal cardiac function.     

Structure of short thick filaments from Limulus muscle

Shortened Limulus thick filaments, isolated from stimulated muscle, are structurally similar to long filaments, isolated from unstimulated muscle, except for length. Both have 3-fold screw symmetry with a helical repeat at approximately 43 nm, axial spacing of 14.5 nm between successive crowns of crossbridges and 4-fold rotational symmetry as estimated from the Bessel argument, by analysis of optical transforms of electron micrograph negatives of negatively stained samples. Both short and long filaments also have similar radii for the location of their crossbridges, thus similar diameters. Equal numbers of subunits/helical strand are also apparent on images of metal-shadowed long and short filaments. Since these data argue against molecular reorganization during filament shortening, it is suggested that the change in length of Limulus thick filaments may occur by reversible disaggregation of constituent protein molecules.     

Hydrostatic pressure studies of native and synthetic thick filaments: II. Native thick filaments from rabbit skeletal muscle

Native thick filaments isolated from freshly prepared rabbit psoas muscle were found to be resistant to pressure-induced dissociation. With increasing pressure application and release, a bimodal distribution of filament lengths was observed. The shorter filament length is associated with filament breakage at the center of the bare zone, while the longer length is associated with relatively intact filaments. Intact filaments and filament halves decrease in length by no more than 20% after exposure to and release of 14,000 psi. Bimodal distributions were not observed in equivalent experiments performed on filaments isolated from muscle glycerinated and stored at -20 degrees C for 6 months. Instead, filament dissociation proceeds linearly as a function of increasing pressure. Filaments prepared from muscle glycerinated and stored for 2 and 4 months exhibited pressure-induced behavior intermediate between the filaments prepared from fresh muscle and filaments prepared from muscle stored for 6 months. Since there appears to be no difference in the protein profiles of the various muscle samples, it is possible that stabilization of the native thick filament against hydrostatic pressure arises from trapped ions that are leached out over time.     

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.