Pradeep K. Luther

Structural Evidence for the Interaction of C-protein (MyBP-C) with Actin and Sequence Identification of a Possible Actin-binding Domain

C-protein (MyBP-C) is a myosin-binding protein that is usually seen in two sets of seven to nine positions in the C-zones in each half of the vertebrate striated muscle A-band. Skeletal muscle C-protein is a modular structure containing ten sub-domains (C1 to C10) of which seven are immunoglobulin-type domains and three (C6, C7 and C9) are fibronectin-like domains. Cardiac muscle C-protein has an extra N-terminal domain (C0) and also some sequence insertions, one of which provides phosphorylation sites. It is conceivable that C-protein has both a structural and regulatory role within the sarcomere. The precise mode of binding of C-protein to the myosin filament has not been determined. However, detailed ultrastructural studies have suggested that C-protein, which binds to myosin, can give rise to a longer periodicity (about 435A) than the intrinsic myosin filament repeat of 429A. The reason for this has remained a puzzle for over 25 years. Here we show by modelling and computation that the presence of this longer periodicity could be explained if the myosin-binding part of C-protein binds to myosin with the expected 429A repeat, but if there are systematic interactions of the N-terminal end of C-protein with the neighbouring actin filaments in the hexagonal lattice of filaments in the A-band. We also show that if they occur these interactions would probably only arise in defined muscle states. Further analysis of the MyBP-C sequence identifies a possible actin-binding domain in the Pro-Ala-rich sequence found at the N terminus of skeletal MyBP-C and between domains C0 and C1 in the cardiac sequence.

Functional improvement and maturation of rat and human engineered heart tissue by chronic electrical stimulation

Spontaneously beating engineered heart tissue (EHT) represents an advanced in vitro model for drug testing and disease modeling, but cardiomyocytes in EHTs are less mature and generate lower forces than in the adult heart. We devised a novel pacing system integrated in a setup for videooptical recording of EHT contractile function over time and investigated whether sustained electrical field stimulation improved EHT properties. EHTs were generated from neonatal rat heart cells (rEHT, n=96) or human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hEHT, n=19). Pacing with biphasic pulses was initiated on day 4 of culture. REHT continuously paced for 16-18 days at 0.5Hz developed 2.2× higher forces than nonstimulated rEHT. This was reflected by higher cardiomyocyte density in the center of EHTs, increased connexin-43 abundance as investigated by two-photon microscopy and remarkably improved sarcomere ultrastructure including regular M-bands. Further signs of tissue maturation include a rightward shift (to more physiological values) of the Ca(2+)-response curve, increased force response to isoprenaline and decreased spontaneous beating activity. Human EHTs stimulated at 2Hz in the first week and 1.5Hz thereafter developed 1.5× higher forces than nonstimulated hEHT on day 14, an ameliorated muscular network of longitudinally oriented cardiomyocytes and a higher cytoplasm-to-nucleus ratio. Taken together, continuous pacing improved structural and functional properties of rEHTs and hEHTs to an unprecedented level. Electrical stimulation appears to be an important step toward the generation of fully mature EHT.

Three-dimensional structure of the vertebrate muscle A-band: III. M-region structure and myosin filament symmetry☆

Ultrathin transverse sections of the body muscle of bony fish and the sartorius muscle of frog have been analysed in detail by optical diffraction and image averaging to reveal the ultrastructures of the myosin filaments both in the M-region and in the outer ends of the filament (the tip region). The evidence is unequivocal that the myosin filaments in both muscle types have 3-fold rotational symmetry in all of the regions where symmetry can be seen. Using the nomenclature of Sjostrom & Squire (1977a), the appearances of cross-sections in fish at different axial locations are as follows. (Note that fish myosin filaments are arranged in a simple hexagonal lattice.) 1. (1)At M1 (central M-bridge line) the myosin filament profile is almost circular, but does sometimes show a three-component structure. There are six M-bridges from each filament. 2. (2) At M4 the filament backbone comprises three kidney-shaped subunits related by a triad axis, together with six M-bridges. There are two types of M-bridge interaction site. 3. (3) At M6 there are three kidney-shaped subunits related by a triad. There are no M-bridges but there does seem to be extra protein here. 4. (4) In the bare region there are three nearly circular subunits related by a triad. 5. (5) At M9 or the start of the bridge region there is a Y-shaped filament profile from which three projections radiate towards three of the six actin filament positions (trigonal points). 6. (6) There is an apparent change of hand of the filament profiles across the M-band when viewed in the same section (i.e. from M4 to M4′ or M6 to M6′). This implies that the bipolar myosin filament has the symmetry of the dihedral point group 32. 7. (7) There is a change in orientation of the 3-fold myosin filament profile across the M-band so that the bare region profiles are about 40 ° apart. Less detail has been seen in frog sartorius muscle sections because of the presence of the superlattice (see paper II in this series, Luther & Squire, 1980). However, the observations are entirely compatible with a myosin filament with the same structure as in fish but arranged in a superlattice. The superlattice is apparent not only in the bare region in frog, but also at the M-bridge level M4. There are two classes of M-bridge interaction at M4 just as in fish. At the A-band edges of both muscles (outer D-zone) the filament profiles appear either triangular, Y-shaped or composed of three subunits. The lattices are insufficiently ordered for satisfactory image averaging to be carried out. As yet the main part of the bridge region shows little ultrastructural detail. However, the present results show clearly that the vertebrate myosin filament has 3-fold rotational symmetry; there can be little doubt that the symmetry of the crossbridge array approximates to that of a three-stranded helix (Squire, 1972). A model is proposed for the structure of the myosin filament in the vertebrate M-region. It explains the observed appearances without going into molecular detail. It is also suggested that it is the M-bridges at M4 that are primarily responsible for defining the two types of A-band structure (simple lattice and superlattice) and that the M1 bridges may have a secondary role.

Three-dimensional structure of the vertebrate muscle A-band: II. The myosin filament superlattice☆☆☆

The three-dimensional arrangement of the myosin filaments in the A-band of frog sartorius muscle was studied using electron micrographs of very thin and accurately cut transverse sections through the bare region (on each side of the M-band) where the thick filament shafts are roughly triangular in shape. It was found that the orientations of these triangular profiles are arranged to give a superlattice of the same size and shape as that proposed by Huxley & Brown (1967) on the basis of X-ray diffraction evidence, but the contents of the superlattice may not be as they suggested. The results from detailed image analysis strongly suggest that myosin filaments (which have been shown to have 3-fold rotational symmetry, Luther, 1978; Luther, Munro and Squire, unpublished results) are arranged with one of two orientations which are 60 ° (or 180 °) apart. This arrangement of filaments with 3-fold symmetry is not that predicted for a superlattice with the symmetry suggested by Huxley & Brown. Two rules define the way in which the orientations of neighbouring filaments are defined. Rule (1): no three mutually adjacent filaments in the hexagonal array of filaments in the A-band can all have identical orientations; and rule (2): no three successive filaments along a 101 row in the filament array can have identical orientations. These two no-three-alike rules are sufficient to describe the observed arrangement of filament profiles in the frog bare region (except for some minor violations discussed in the text), and they lead automatically to the generation of the required superlattice. The A-band structure in fish muscle is different; there is no superlattice and the triangular bare region profiles have only one orientation. The frog superlattice and fish simple lattice are explained directly in terms of different interactions between the M-bridges in the M-bands of these muscles. The observed structures require that the myosin filament symmetry at the centre of the M-band is that of the dihedral point group 32. The two possible forms of interaction between filaments with this symmetry (apart from a completely random structure) give rise to the observed A-band lattices in frog and fish muscles. The 3-fold rotational symmetry of the myosin filaments required to explain the observed micrographs also requires that the myosin crossbridge arrangements around the actin filaments in frog and fish muscles will be different. It is suggested that the structure in the frog A-band (and in the A-bands of other higher vertebrates) has evolved from that in fish to improve the distribution of crossbridges around the aotin filaments. The X-ray diffraction evidence of Huxley & Brown (1967) will be accounted for in terms of the proposed A-band structure in a further paper in this series.

Three-dimensional structure of the vertebrate muscle M-region

Abstract By applying accurate sectioning methods to well-ordered muscles obtained using “ in situ ” fixation, new details of the structure of the M-band and M-region of frog sartorius muscle have been obtained. Results are described which support the three-dimensional M-band model proposed by Knappeis & Carlsen (1968) but which conflict with the model proposed by Pepe (1975). In addition, new bridge-like structures have been observed both in the M-band (in addition to the well-known M-bridges) and at the edge of the M-region (pseudo H-zone). Finally, it is noted that all of the results presented here provide support for the idea that the myosin crossbridges in this muscle are arranged on a three-stranded helix.

Direct visualization of myosin-binding protein C bridging myosin and actin filaments in intact muscle

Myosin-binding protein C (MyBP-C) is a thick filament protein playing an essential role in muscle contraction, and MyBP-C mutations cause heart and skeletal muscle disease in millions worldwide. Despite its discovery 40 y ago, the mechanism of MyBP-C function remains unknown. In vitro studies suggest that MyBP-C could regulate contraction in a unique way—by bridging thick and thin filaments—but there has been no evidence for this in vivo. Here we use electron tomography of exceptionally well preserved muscle to demonstrate that MyBP-C does indeed bind to actin in intact muscle. This binding implies a physical mechanism for communicating the relative sliding between thick and thin filaments that does not involve myosin and which could modulate the contractile process.

Understanding the Organisation and Role of Myosin Binding Protein C in Normal Striated Muscle by Comparison with MyBP-C Knockout Cardiac Muscle

Myosin binding protein C (MyBP-C) is a component of the thick filament of striated muscle. The importance of this protein is revealed by recent evidence that mutations in the cardiac gene are a major cause of familial hypertrophic cardiomyopathy. Here we investigate the distribution of MyBP-C in the A-bands of cardiac and skeletal muscles and compare this to the A-band structure in cardiac muscle of MyBP-C-deficient mice. We have used a novel averaging technique to obtain the axial density distribution of A-bands in electron micrographs of well-preserved specimens. We show that cardiac and skeletal A-bands are very similar, with a length of 1.58 ± 0.01 μm. In normal cardiac and skeletal muscle, the distributions are very similar, showing clearly the series of 11 prominent accessory protein stripes in each half of the A-band spaced axially at 43-nm intervals and starting at the edge of the bare zone. We show by antibody labelling that in cardiac muscle the distal nine stripes are the location of MyBP-C. These stripes are considerably suppressed in the knockout mouse hearts as expected. Myosin heads on the surface of the thick filament in relaxed muscle are thought to be arranged in a three-stranded quasi-helix with a mean 14.3-nm axial cross bridge spacing and a 43 nm helix repeat. Extra “forbidden” meridional reflections, at orders of 43 nm, in X-ray diffraction patterns of muscle have been interpreted as due to an axial perturbation of some levels of myosin heads. However, in the MyBP-C-deficient hearts these extra meridional reflections are weak or absent, suggesting that they are due to MyBP-C itself or to MyBP-C in combination with a head perturbation brought about by the presence of MyBP-C.

Muscle ultrastructure in the teleost fish

Abstract The muscles of teleost fish are different in ultrastructure from those of most other kinds of vertebrate. A distinct difference is that they are much more highly ordered than the frog, rabbit or human muscles conventionally used as vertebrate muscle stereotypes. The improved order permits the full application of powerful techniques such as electron microscopy and image processing, and X-ray diffraction and model building. Here ultrastructural aspects of the red and white fibres in typical teleosts are compared through paired sets of electron micrographs, and ultrastructural details of teleost M-bands and Z-bands obtained from the application of three-dimensional reconstruction methods are described. The potential application of teleost muscle in production of “Muscle—The Movie”, which it is hoped will help to define the mechanism of force generation, is introduced.

M-band structure, M-bridge interactions and contraction speed in vertebrate cardiac muscles

SummaryCardiac muscle M-band structures in several mammals (guinea pig, rabbit, rat and cow) and also from three teleosts (plaice, carp and roach), have been studied using electron microscopy and image processing. Axial structure seen in negatively stained isolated myofibrils or negatively stained cryo-sections shows the presence of five strong M-bridge lines (M6, M4, M1, M4′ and M6′) except in the case of the teleost M-bands in which the central M-line (M1) is absent, giving a four-line M-band. The M4 (M4′) lines are consistently strong in all muscles, supporting the suggestion that bridges at this position are important for the structural integrity of the A-band myosin filament lattice. Across the vertebrate kingdom, cardiac M-band ultrastructure appears to correlate roughly with heartbeat frequency, just as in skeletal muscles it correlates with contraction speed, reinforcing the suggestion that some M-band components may have a significant physiological role. Apart from rat heart, which is relatively fast and has a conventional five-line M-band with M1 and M4 approximately equal, the rabbit, guinea pig and beef heart M-bands form a new 1+4 class; M1 is relatively very much stronger than M4.Transverse sections of the teleost (roach) cardiac A-band show a simple lattice arrangement of myosin filaments, just as teleost skeletal muscles. Almost all other vertebrate striated muscles, including mammalian heart muscles, have a statistical superlattice structure. The high degree of filament lattice order in teleost cardiac muscles indicates their potential usefulness for ultrastructural studies.It is shown that, in four-line M-bands in which the central (M1) M-bridges are missing, interactions at M4 (M4′) are sufficient to define the different myosin filament orientations in simple lattice and superlattice A-bands. However the presence of M1 bridges may improve the axial order of the A-band.

Muscle Z-band ultrastructure: titin Z-repeats and Z-band periodicities do not match.

Vertebrate muscle Z-bands show zig-zag densities due to different sets of alpha-actinin cross-links between anti-parallel actin molecules. Their axial extent varies with muscle and fibre type: approximately 50 nm in fast and approximately 100 nm in cardiac and slow muscles, corresponding to the number of alpha-actinin cross-links present. Fish white (fast) muscle Z-bands have two sets of alpha-actinin links, mammalian slow muscle Z-bands have six. The modular structure of the approximately 3 MDa protein titin that spans from M-band to Z-band correlates with the axial structure of the sarcomere; it may form the template for myofibril assembly. The Z-band-located amino-terminal 80 kDa of titin includes 45 residue repeating modules (Z-repeats) that are expressed differentially; heart, slow and fast muscles have seven, four to six and two to four Z-repeats, respectively. Gautel et al. proposed a Z-band model in which each Z-repeat links to one level of alpha-actinin cross-links, requiring that the axial extent of a Z-repeat is the same as the axial separation of alpha-actinin layers, of which there are two in every actin crossover repeat. The span of a Z-repeat in vitro is estimated by Atkinson et al. to be 12 nm or less; much less than half the normal vertebrate muscle actin crossover length of 36 nm. Different actin-binding proteins can change this length; it is reduced markedly by cofilin binding, or can increase to 38.5 nm in the abnormally large nemaline myopathy Z-band. Here, we tested whether in normal vertebrate Z-bands there is a marked reduction in crossover repeat so that it matches twice the apparent Z-repeat length of 12 nm. We found that the measured periodicities in wide Z-bands in slow and cardiac muscles are all very similar, about 39 nm, just like the nemaline myopathy Z-bands. Hence, the 39 nm periodicity is an important conserved feature of Z-bands and either cannot be explained by titin Z-repeats as previously suggested or may correlate with two Z-repeats.