Cardiac Myosin-binding protein C modulates the tuning of the molecular motor in the heart

Cardiac myosin binding protein C (cMyBP-C) is an important regulator of cardiac contractility. Its precise effect on myosin cross-bridges (CBs) remains unclear. Using a cMyBP-C^−/− mouse model, we determined how cMyBP-C modulates the cyclic interaction of CBs with actin. From papillary muscle mechanics, CB characteristics were provided using A. F. Huxley's equations. The probability of myosin being weakly bound to actin was higher in cMyBP-C^−/− than in cMyBP-C^+/+. However, the number of CBs in strongly bound, high-force generated state and the force generated per CB were lower in cMyBP-C^−/−. Overall CB cycling and the velocity of CB tilting were accelerated in cMyBP-C^−/−. Taking advantage of the presence of cMyBP-C in cMyBP-C^+/+ myosin solution but not in cMyBP-C^−/−, we also analyzed the effects of cMyBP-C on the myosin-based sliding velocity of actin filaments. At baseline, sliding velocity and the relative isometric CB force, as determined by the amount of α-actinin required to arrest thin filament motility, were lower in cMyBP-C^−/− than in cMyBP-C^+/+. cAMP-dependent protein kinase-mediated cMyBP-C phosphorylation further increased the force produced by CBs. We conclude that cMyBP-C prevents inefficient, weak binding of the myosin CB to actin and has a critical effect on the power-stroke step of the myosin molecular motor.     

Cardiac Myosin Binding Protein-C Is Essential for Thick-Filament Stability and Flexural Rigidity

Using atomic force microscopy, we examined the contribution of cardiac myosin binding protein-C (cMyBP-C) to thick-filament length and flexural rigidity. Native thick filaments were isolated from the hearts of transgenic mice bearing a truncation mutation of cMyBP-C (t/t) that results in no detectable cMyBP-C and from age-matched wild-type controls (+/+). Atomic force microscopy images of these filaments were evaluated with an automated analysis algorithm that identified filament position and shape. The t/t thick-filament length (1.48 ± 0.02 μm) was significantly (P < 0.01) shorter than +/+ (1.56 ± 0.02 μm). This 5%-shorter thick-filament length in the t/t was reflected in 4% significantly shorter sarcomere lengths of relaxed isolated cardiomyocytes of the t/t (1.97 ± 0.01 μm) compared to +/+ (2.05 ± 0.01 μm). To determine if cMyBP-C contributes to the mechanical properties of thick filaments, we used statistical polymer chain mechanics to calculate a per-filament-specific persistence length, an index of flexural rigidity directly proportional to Young's modulus. Thick-filament-specific persistence length in the t/t (373 ± 62 μm) was significantly lower than in +/+ (639 ± 101 μm). Accordingly, Young's modulus of t/t thick filaments was ∼60% of +/+. These results provide what we consider a new understanding for the critical role of cMyBP-C in defining normal cardiac output by sustaining force and muscle stiffness.     

The vertebrate skeletal muscle thick filaments are not three-stranded. Reinterpretation of some experimental data

Computer simulation of mass distribution within the model and Fourier transforms of images depicting mass distribution are explored for verification of two alternative modes of the myosin molecule arrangement within the vertebrate skeletal muscle thick filaments. The model well depicting the complete bipolar structure of the thick filament and revealing a true threefold-rotational symmetry is a tube covered by two helices with a pitch of 2 x 43 nm due to arrangement of the myosin tails along a helical path and grouping of all myosin heads in the crowns rotated by 240 degrees and each containing three cross-bridges separated by 0 degrees, 120 degrees, and 180 degrees. The cross-bridge crown parameters are verified by EM images as well as by optical and low-angle X-ray diffraction patterns found in the literature. The myosin tail arrangement, at which the C-terminus of about 43-nm length is near-parallel to the filament axis and the rest of the tail is quite strongly twisted around, is verified by the high-angle X-ray diffraction patterns. A consequence of the new packing is a new way of movement of the myosin cross-bridges, namely, not by bending in the hinge domains, but by unwrapping from the thick filament surface towards the thin filaments along a helical path.     

Cryo-Electron Microscopy Reveals Cardiac Myosin Binding Protein-C M-Domain Interactions with the Thin Filament

Cardiac myosin binding protein C (cMyBP-C) modulates cardiac contraction via direct interactions with cardiac thick (myosin) and thin (actin) filaments (cTFs). While its C-terminal domains (e.g. C8-C10) anchor cMyBP-C to the backbone of the thick filament, its N-terminal domains (NTDs) (e.g. C0, C1, M, and C2) bind to both myosin and actin to accomplish its dual roles of inhibiting thick filaments and activating cTFs. While the positions of C0, C1 and C2 on cTF have been reported, the binding site of the M-domain on the surface of the cTF is unknown. Here, we used cryo-EM to reveal that the M-domain interacts with actin via helix 3 of its ordered tri-helix bundle region, while the unstructured part of the M-domain does not maintain extensive interactions with actin. We combined the recently obtained structure of the cTF with the positions of all the four NTDs on its surface to propose a complete model of the NTD binding to the cTF. The model predicts that the interactions of the NTDs with the cTF depend on the activation state of the cTF. At the peak of systole, when bound to the extensively activated cTF, NTDs would inhibit actomyosin interactions. In contrast, at falling Ca²⁺ levels, NTDs would not compete with the myosin heads for binding to the cTF, but would rather promote formation of active cross-bridges at the adjacent regulatory units located at the opposite cTF strand. Our structural data provides a testable model of the cTF regulation by the cMyBP-C.     

Radial displacement of myosin cross-bridges in mouse myocardium due to ablation of myosin binding protein-C

Myosin binding protein-C (cMyBP-C) is a thick filament accessory protein, which in cardiac muscle functions to regulate the kinetics of cross-bridge interaction with actin; however, the underlying mechanism is not yet understood. To explore the structural basis for cMyBP-C function, we used synchrotron low-angle X-ray diffraction to measure interfilament lattice spacing and the equatorial intensity ratio, I(11)/I(10), in skinned myocardial preparations isolated from wild-type (WT) and cMyBP-C null (cMyBP-C(-/-)). In relaxed myocardium, ablation of cMyBP-C appeared to result in radial displacement of cross-bridges away from the thick filaments, as there was a significant increase ( approximately 30%) in the I(11)/I(10) ratio for cMyBP-C(-/-) (0.37+/-0.03) myocardium as compared to WT (0.28+/-0.01). While lattice spacing tended to be greater in cMyBP-C(-/-) myocardium (44.18+/-0.68 nm) when compared to WT (42.95+/-0.43 nm), the difference was not statistically significant. Furthermore, liquid-like disorder in the myofilament lattice was significantly greater ( approximately 40% greater) in cMyBP-C(-/-) myocardium as compared to WT. These results are consistent with our working hypothesis that cMyBP-C normally acts to tether myosin cross-bridges nearer to the thick filament backbone, thereby reducing the likelihood of cross-bridge binding to actin and limiting cooperative activation of the thin filament.     

Multiple forms of cardiac myosin-binding protein C exist and can regulate thick filament stability

Although absence or abnormality of cardiac myosin binding protein C (cMyBP-C) produces serious structural and functional abnormalities of the heart, function of the protein itself is not clearly understood, and the cause of the abnormalities, unidentified. Here we report that a major function of cMyBP-C may be regulating the stability of the myosin-containing contractile filaments through phosphorylation of cMyBP-C. Antibodies were raised against three different regions of cMyBP-C to detect changes in structure within the molecule, and loss of myosin heavy chain was used to monitor degradation of the thick filament. Results from Western blotting and polyacrylamide gel electrophoresis indicate that cMyBP-C can exist in two different forms that produce, respectively, stable and unstable thick filaments. The stable form has well-ordered myosin heads and requires phosphorylation of the cMyBP-C. The unstable form has disordered myosin heads. In tissue with intact cardiac cells, the unstable unphosphorylated cMyBP-C is more easily proteolyzed, causing thick filaments first to release cMyBP-C and/or its proteolytic peptides and then myosin. Filaments deficient in cMyBP-C are fragmented by shear force well tolerated by the stable form. We hypothesize that modulation of filament stability can be coupled at the molecular level with the strength of contraction by the sensitivity of each to the concentration of calcium ions.     

Recent X-ray Diffraction and Electron Microscope Studies of Striated Muscle

The sliding filament model for muscular contraction supposes that an appropriately directed force is developed between the actin and myosin filaments by some process in which the cross-bridges are involved. The cross-bridges between the filaments are believed to represent the parts of the myosin molecules which possess the active sites for ATPase activity and actin-binding ability, and project out sidewise from the backbone of the thick filaments. The arrangement of the cross-bridges is now being studied by improved low-angle X-ray diffraction techniques, which show that in a resting muscle, they are arranged approximately but not exactly in a helical pattern, and that there are other structural features of the thick filaments which give rise to additional long periodicities shown up by the X-ray diagram. The actin filaments also contain helically arranged subunits, and both the subunit repeat and the helical repeat are different from those in the myosin filaments. Diffraction diagrams can be obtained from muscles in rigor (when permanent attachment of the cross-bridges to the actin subunits takes place) and now, taking advantage of the great increase in the speed of recording, from actively contracting muscles. These show that changes in the arrangement of the cross-bridges are produced under both these conditions and are no doubt associated in contraction with the development of force. Thus configurational changes of the myosin component in muscle have been demonstrated: these take place without any significant over-all change in the length of the filaments.     

AN ultrastructural study of cross-bridge arrangement in the frog thigh muscle thick filament.

We have developed thick filament isolation methods that preserve the relaxed cross-bridge order of frog thick filaments such that the filaments can be analyzed by the convergent techniques of electron microscopy, optical diffraction, and computer image analysis. Images of the filaments shadowed by using either unidirectional shadowing or rotary shadowing show a series of subunits arranged along a series of right-handed near-helical strands that occur every 43 nm axially along the filament arms. Optical filtrations of images of these shadowed filaments show 4-5 subunits per half-turn of the strands, consistent with a three-stranded arrangement of the cross-bridges, thus supporting our earlier results from negative staining and computer-image analysis. The optical diffraction patterns of the shadowed filaments show a departure from the pattern expected for helical symmetry consistent with the presence of cylindrical symmetry and a departure of the cross-bridges from helical symmetry. We also describe a modified negative staining procedure that gives improved delineation of the cross-bridge arrangement. From analysis of micrographs of these negatively stained filament tilted about their long axes, we have computed a preliminary three-dimensional reconstruction of the filament that clearly confirms the three-stranded arrangement of the myosin heads.     

Oblique section 3-D reconstruction of relaxed insect flight muscle reveals the cross-bridge lattice in helical registration.

In this work we examined the arrangement of cross-bridges on the surface of myosin filaments in the A-band of Lethocerus flight muscle. Muscle fibers were fixed using the tannic-acid-uranyl-acetate, ("TAURAC") procedure. This new procedure provides remarkably good preservation of native features in relaxed insect flight muscle. We computed 3-D reconstructions from single images of oblique transverse sections. The reconstructions reveal a square profile of the averaged myosin filaments in cross section view, resulting from the symmetrical arrangement of four pairs of myosin heads in each 14.5-nm repeat along the filament. The square profiles form a very regular right-handed helical arrangement along the surface of the myosin filament. Furthermore, TAURAC fixation traps a near complete 38.7 nm labeling of the thin filaments in relaxed muscle marking the left-handed helix of actin targets surrounding the thick filaments. These features observed in an averaged reconstruction encompassing nearly an entire myofibril indicate that the myosin heads, even in relaxed muscle, are in excellent helical register in the A-band.     

Disorder induced in nonoverlap myosin cross-bridges by loss of adenosine triphosphate.

Adenosine triphosphate-dependent changes in myosin filament structure have been directly observed in whole muscle by electron microscopy of thin sections of rapidly frozen, demembranated frog sartorius specimens. In the presence of ATP the thick filaments show an ordered, helical array of cross-bridges except in the bare zone. In the absence of ATP they show two distinct appearances: in the region of overlap with actin, there is an ordered, rigorlike array of cross-bridges between the thick and thin filaments, whereas in the nonoverlap region (H-zone) the myosin heads move away from the thick filament backbone and lose their helical order. This result suggests that the presence of ATP is necessary for maintenance of the helical array of cross-bridges characteristic of the relaxed state. The primary effect of ATP removal on the myosin heads appears to be weaken their binding to the thick filament backbone; released heads that are close to an actin filament subsequently form a new actin-based, ordered array.     

Structure and paramyosin content of tarantula thick filaments

Muscle fibers of the tarantula femur exhibit structural and biochemical characteristics similar to those of other long-sarcomere invertebrate muscles, having long A-bands and long thick filaments. 9-12 thin filaments surround each thick filament. Tarantula muscle has a paramyosin:myosin heavy chain molecular ratio of 0.31 +/- 0.079 SD. We studied the myosin cross-bridge arrangement on the surface of tarantula thick filaments on isolated, negatively stained, and unidirectionally metal-shadowed specimens by electron microscopy and optical diffraction and filtering and found it to be similar to that previously described for the thick filaments of muscle of the closely related chelicerate arthropod, Limulus. Cross-bridges are disposed in a four-stranded right-handed helical arrangement, with 14.5-nm axial spacing between successive levels of four bridges, and a helical repeat period every 43.5 nm. The orientation of cross-bridges on the surface of tarantula filaments is also likely to be very similar to that on Limulus filaments as suggested by the similarity between filtered images of the two types of filaments and the radial distance of the centers of mass of the cross-bridges from the surfaces of both types of filaments. Tarantula filaments, however, have smaller diameters than Limulus filaments, contain less paramyosin, and display structure that probably reflects the organization of the filament backbone which is not as apparent in images of Limulus filaments. We suggest that the similarities between Limulus and tarantula thick filaments may be governed, in part, by the close evolutionary relationship of the two species.     

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.     

Multiple structures of thick filaments in resting cardiac muscle and their influence on cross-bridge interactions.

Based on two criteria, the tightness of packing of myosin rods within the backbone of the filament and the degree of order of the myosin heads, thick filaments isolated from a control group of rat hearts had three different structures. Two of the structures of thick filaments had ordered myosin heads and were distinguishable from each other by the difference in tightness of packing of the myosin rods. Depending on the packing, their structure has been called loose or tight. The third structure had narrow shafts and disordered myosin heads extending at different angles from the backbone. This structure has been called disordered. After phosphorylation of myosin-binding protein C (MyBP-C) with protein kinase A (PKA), almost all thick filaments exhibited the loose structure. Transitions from one structure to another in quiescent muscles were produced by changing the concentration of extracellular Ca. The probability of interaction between isolated thick and thin filaments in control, PKA-treated preparations, and preparations exposed to different Ca concentrations was estimated by electron microscopy. Interactions were more frequent with phosphorylated thick filaments having the loose structure than with either the tight or disordered structure. In view of the presence of MgATP and the absence of Ca, the interaction between the myosin heads and the thin filaments was most likely the weak attachment that precedes the force-generating steps in the cross-bridge cycle. These results suggest that phosphorylation of MyBP-C in cardiac thick filaments increases the probability of cross-bridges forming weak attachments to thin filaments in the absence of activation. This mechanism may modulate the number of cross-bridges generating force during activation.     

A Hypertrophic Cardiomyopathy-associated MYBPC3 Mutation Common in Populations of South Asian Descent Causes Contractile Dysfunction

Hypertrophic cardiomyopathy (HCM) results from mutations in genes encoding sarcomeric proteins, most often MYBPC3, which encodes cardiac myosin binding protein-C (cMyBP-C). A recently discovered HCM-associated 25-base pair deletion in MYBPC3 is inherited in millions worldwide. Although this mutation causes changes in the C10 domain of cMyBP-C (cMyBP-C^C10mut), which binds to the light meromyosin (LMM) region of the myosin heavy chain, the underlying molecular mechanism causing HCM is unknown. In this study, adenoviral expression of cMyBP-C^C10mut in cultured adult rat cardiomyocytes was used to investigate protein localization and evaluate contractile function and Ca²⁺ transients, compared with wild-type cMyBP-C expression (cMyBP-C^WT) and controls. Forty-eight hours after infection, 44% of cMyBP-C^WT and 36% of cMyBP-C^C10mut protein levels were determined in total lysates, confirming equal expression. Immunofluorescence experiments showed little or no localization of cMyBP-C^C10mut to the C-zone, whereas cMyBP-C^WT mostly showed C-zone staining, suggesting that cMyBP-C^C10mut could not properly integrate in the C-zone of the sarcomere. Subcellular fractionation confirmed that most cMyBP-C^C10mut resided in the soluble fraction, with reduced presence in the myofilament fraction. Also, cMyBP-C^C10mut displayed significantly reduced fractional shortening, sarcomere shortening, and relaxation velocities, apparently caused by defects in sarcomere function, because Ca²⁺ transients were unaffected. Co-sedimentation and protein cross-linking assays confirmed that C10^mut causes the loss of C10 domain interaction with myosin LMM. Protein homology modeling studies showed significant structural perturbation in cMyBP-C^C10mut, providing a potential structural basis for the alteration in its mode of interaction with myosin LMM. Therefore, expression of cMyBP-C^C10mut protein is sufficient to cause contractile dysfunction in vitro.     

Electron microscopic and optical diffraction analysis of the structure of scorpion muscle thick filaments

We rapidly and gently isolated thick filaments from scorpion tail muscle by a modification of the technique previously described for isolating Limulus thick filaments. Images of negatively stained filaments appeared to be highly periodic, with a well-preserved myosin cross-bridge array. Optical diffraction patterns of the electron micrograph images were detailed and similar to optical diffraction patterns from Limulus and tarantula thick filaments. Analysis of the optical diffraction patterns and computed Fourier transforms, together with the appearance of the filaments in the micrographs, suggested a model for the filaments in which the myosin cross-bridges were arranged on four helical strands with 12 cross-bridges per turn of each strand, thus giving the observed repeat every third cross-bridge level. Comparison of the scorpion thick filaments with those isolated from the closely related chelicerate arthropods, Limulus and tarantula, revealed that they were remarkably similar in appearance and helical symmetry but different in diameter.     

Nanosurfer assay dissects β-cardiac myosin and cardiac myosin-binding protein C interactions

Cardiac myosin-binding protein C (cMyBP-C) modulates cardiac contractility through putative interactions with the myosin S2 tail and/or the thin filament. The relative contribution of these binding-partner interactions to cMyBP-C modulatory function remains unclear. Hence, we developed a “nanosurfer” assay as a model system to interrogate these cMyBP-C binding-partner interactions. Synthetic thick filaments were generated using recombinant human β-cardiac myosin subfragments (HMM or S1) attached to DNA nanotubes, with 14- or 28-nm spacing, corresponding to the 14.3-nm myosin spacing in native thick filaments. The nanosurfer assay consists of DNA nanotubes added to the in vitro motility assay so that myosins on the motility surface effectively deliver thin filaments to the DNA nanotubes, enhancing thin filament gliding probability on the DNA nanotubes. Thin filament velocities on nanotubes with either 14- or 28-nm myosin spacing were no different. We then characterized the effects of cMyBP-C on thin filament motility by alternating HMM and cMyBP-C N-terminal fragments (C0–C2 or C1–C2) on nanotubes every 14 nm. Both C0–C2 and C1–C2 reduced thin filament velocity four- to sixfold relative to HMM alone. Similar inhibition occurred using the myosin S1 construct, which lacks the myosin S2 region proposed to interact with cMyBP-C, suggesting that the cMyBP-C N terminus must interact with other myosin head domains and/or actin to slow thin filament velocity. Thin filament velocity was unaffected by the C0–C1f fragment, which lacks the majority of the M-domain, supporting the importance of this domain for inhibitory interaction(s). A C0–C2 fragment with phospho-mimetic replacement in the M-domain showed markedly less inhibition of thin filament velocity compared with its phospho-null counterpart, highlighting the modulatory role of M-domain phosphorylation on cMyBP-C function. Therefore, the nanosurfer assay provides a platform to precisely manipulate spatially dependent cMyBP-C binding-partner interactions, shedding light on the molecular regulation of β-cardiac myosin contractility.     

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.     

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.     

Unfixed cryosections of striated muscle to study dynamic molecular events.

The structures of the actin and myosin filaments of striated muscle have been studied extensively in the past by sectioning of fixed specimens. However, chemical fixation alters molecular details and prevents biochemically induced structural changes. To overcome these problems, we investigate here the potential of cryosectioning unfixed muscle. In cryosections of relaxed, unfixed specimens, individual myosin filaments displayed the characteristic helical organization of detached cross-bridges, but the filament lattice had disintegrated. To preserve both the filament lattice and the molecular structure of the filaments, we decided to section unfixed rigor muscle, stabilized by actomyosin cross-bridges. The best sections showed periodic, angled cross-bridges attached to actin and their Fourier transforms displayed layer lines similar to those in x-ray diffraction patterns of rigor muscle. To preserve relaxed filaments in their original lattice, unfixed sections of rigor muscle were picked up on a grid and relaxed before negative staining. The myosin and actin filaments showed the characteristic helical arrangements of detached cross-bridges and actin subunits, and Fourier transforms were similar to x-ray patterns of relaxed muscle. We conclude that the rigor structure of muscle and the ability of the filament lattice to undergo the rigor-relaxed transformation can be preserved in unfixed cryosections. In the future, it should be possible to carry out dynamic studies of active sacromeres by cryo-electron microscopy.     

Blebbistatin stabilizes the helical order of myosin filaments by promoting the switch 2 closed state

Blebbistatin is a small-molecule, high-affinity, noncompetitive inhibitor of myosin II. We have used negative staining electron microscopy to study the effects of blebbistatin on the organization of the myosin heads on muscle thick filaments. Loss of ADP and Pi from the heads causes thick filaments to lose their helical ordering. In the presence of 100 μM blebbistatin, disordering was at least 10 times slower. In the M·ADP state, myosin heads are also disordered. When blebbistatin was added to M·ADP thick filaments, helical ordering was restored. However, blebbistatin did not improve the order of thick filaments lacking bound nucleotide. Addition of calcium to relaxed muscle homogenates induced thick-thin filament interaction and filament sliding. In the presence of blebbistatin, filament interaction was inhibited. These structural observations support the conclusion, based on biochemical studies, that blebbistatin inhibits myosin ATPase and actin interaction by stabilizing the closed switch 2 structure of the myosin head. These properties make blebbistatin a useful tool in structural and functional studies of cell motility and muscle contraction.