Genetic dissection of Drosophila myofibril formation: effects of actin and myosin heavy chain null alleles

We used null mutations of Drosophila actin and myosin genes to investigate two aspects of myofibril assembly. First, we eliminated all actin or myosin in flight muscles to evaluate contributions of thick and thin filaments to sarcomere formation. Results demonstrate that thick and thin filament arrays can assemble independently but that both are essential for sarcomeric order and periodicity. Second, we examined how filament stoichiometry affects myofibril assembly. We find that heterozygotes for actin (Act88F) or myosin heavy chain (Mhc36B) null alleles have complex myofibrillar defects, whereas Mhc36B-/+; Act88F-/+ double heterozygotes have nearly normal myofibrils. These results imply that most defects observed in single heterozygotes are due to filament imbalances, not deficits, and suggest that thick and thin filament interactions regulate myofibrillar growth and alignment.     

Assembly of thick, thin, and titin filaments in chick precardiac explants.

De novo cardiac myofibril assembly has been difficult to study due to the lack of available cell culture models that clearly and accurately reflect heart muscle development in vivo. However, within precardiac chick embryo explants, premyocardial cells differentiate and commence beating in a temporal pattern that corresponds closely with myocyte differentiation in the embryo. Immunofluorescence staining of explants followed by confocal microscopy revealed that distinct stages of cardiac myofibril assembly, ranging from the earliest detection of sarcomeric proteins to the late appearance of mature myofibrils, were consistently recognized in precardiac cultures. Assembly events involved in the early formation of sarcomeres were clearly visualized and accurately reflected observations described by others during chick heart muscle development. Specifically, the early colocalization of alpha-actinin and titin dots was observed near the cell periphery representing I-Z-I-like complex formation. Myosin-containing thick filaments assembled independently of actin-containing thin filaments and appeared centered within sarcomeres when titin was also linearly aligned at or near cell borders. An N-terminal epitope of titin was detected earlier than a C-terminal epitope; however, both epitopes were observed to alternate near the cell periphery concomitant with the earliest formation of myofibrils. Although vascular actin was detected within cells during early assembly stages, cardiac actin predominated as the major actin isoform in mature thin filaments. Well-aligned thin filaments were also observed in the absence of organized staining for tropomodulin at thin filament pointed ends, suggesting that tropomodulin is not required to define thin filament lengths. Based on these findings, we conclude that the use of the avian precardiac explant system accurately allows for direct investigation of the mechanisms regulating de novo cardiac myofibrillogenesis.     

High-voltage electron microscopy of crossbridge interactions in striated muscle

Summary In order to investigate the geometry of the interactions which myosin molecules make with actin filaments we have studied thick (0.2–0.5 µm) transverse sections of striated muscles in the 1 MeV electron microscope at Imperial College. Sections obtained from fixed relaxed frog sartorius muscle and both fixed relaxed and fixed rigor insect flight muscles, show regular electron opaque features between the thick and thin filament profiles. These are thought to be the overlapping images of the many levels of myosin heads that occur in such sections. From the appearances of these images, together with studies of thin transverse sections, it appears that of the possible interactions which one myosin molecule can make, namely that its two component heads interact with the same thin filament or with two different thin filaments, it is the former interaction (both heads on the same filament) which is predominant. Nevertheless appearances have been seen similar to those expected if an interaction of one molecule with two thin filaments occurs. It is concluded that both single filament and two filament interactions can occur depending on the steric convenience of the available actin subunits, but that the single filament interaction occurs in the majority of cases in the muscle states we have studied.Finally it is shown that the myosin filament profiles seen in thick transverse sections may be a very misleading guide to thick filament structure because of the influence which the myosin crossbridges have on the appearance of the profiles.     

Initiation of apoptosis by actin cytoskeletal derangement in human airway epithelial cells

Changes in epithelial cell shape can lead to cell death and detachment. Actin filaments are cleaved during apoptosis, but whether disruption in the actin cytoskeletal network, as one manifestation of cell shape change, can itself induce apoptosis is not known. We tested this hypothesis in the airway epithelial cell line 1HAEo(-) and in primary airway epithelial cells by preventing actin filament elongation with cytochalasin D or by aggregating actin filaments with jasplakinolide. Disruption of actin filament integrity promptly induced apoptosis in adherent epithelial cells within 5 h. Jasplakinolide-induced apoptosis did not disrupt focal adhesions, whereas cytochalasin D-induced apoptosis decreased focal adhesion protein expression and occurred despite ligation of the fibronectin receptor. Death induction was abrogated by the caspase inhibitors z-VAD-fmk and Ac-DEVD-cho but not by blocking the Fas (CD95) receptor. Whereas cytochalasin D--induced apoptosis was associated with cleavage of pro-caspase-8, jasplakinolide-induced apoptosis was not. Both agents induced formation of a death-inducing signaling complex. These data demonstrate that disruption of actin filament integrity with either cytochalasin D or jasplakinolide induces apoptosis in airway epithelial cells but by different mechanisms, and suggest that actin may be an early modulator of apoptotic commitment.     

Geometrical constraints affecting crossbridge formation in insect flight muscle

Summary Computer-modelling studies have explored how rigor crossbridge interactions in insect flight muscle are affected by using a four-stranded helical thick filament and by restricting each myosin to forming one crossbridge with only one actin filament. Crossbridges searching over an axial range of ±7.2 nm, and within an azimuthal range around actin of ±45°, can simulate the actin-labelling patterns observed in thin electron microscope sections well. However, the number of crossbridges attached between any myosin filament and an adjacent actin filament depends on their relative axial and azimuthal positions, and can vary by a factor of two. The relative position that maximized the number of attached bridges also produced the best modelling of the ‘double chevron’ appearance of two crossbridge pairs attaching within target zones every 38.6 nm, as seen in thin longitudinal sections, and the ‘flared X’ of crossbridges extending to four out of six surrounding actins at each crossbridge level seen in thin cross-sections. Micrographs show that excellent lattice register of rigor crossbridges in longitudinal sections does not depend on lateral register of thick or thin filament ends. Our modelling suggests how the crossbridge lattice may be generated by filaments becoming mutually annealed to the axial and azimuthal positions at which most crossbridges can attach, at which time the actin filaments are arranged at the diad positions of the P6₄ crystalline lattice. When the actin filaments are so oriented, in a P6₄ lattice, two crossbridges on adjacent actin filaments will slew toward the same point on the myosin filament, producing the flared X appearance of origin from a common stem and a single myosin, even if they originate from distinct points and separate molecules.     

Structure, interactions and function of the N-terminus of cardiac myosin binding protein C (MyBP-C): who does what, with what, and to whom?

The thick filament protein myosin-binding protein-C shows a highly modular architecture, with the C-terminal region responsible for tethering to the myosin and titin backbone of the thick filament. The N-terminal region shows the most significant differences between cardiac and skeletal muscle isogenes: an entire Ig-domain (C0) is added, together with highly regulated phosphorylation sites between Ig domains C1 and C2. These structural and functional differences at the N-terminus reflect important functions in cardiac muscle regulation in health and disease. Alternative interactions of this part of MyBP-C with the head-tail (S1-S2) junction of myosin or to actin filaments have been proposed, but with conflicting experimental evidence. The regulation of myosin or actin interaction by phosphorylation of the cardiac MyBP-C N-terminus may play an additional role in length-dependent contraction regulation. We discuss here the evidence for these proposed interactions, considering the required properties of MyBP-C, the way in which they may be regulated in muscle contraction and the way they might be related to heart disease. We also attempt to shed some light on experimental pitfalls and future strategies.     

Earning stripes: myosin binding protein-C interactions with actin

Myosin binding protein-C (MyBP-C) was first discovered as an impurity during the purification of myosin from skeletal muscle. However, soon after its discovery, MyBP-C was also shown to bind actin. While the unique functional implications for a protein that could cross-link thick and thin filaments together were immediately recognized, most early research nonetheless focused on interactions of MyBP-C with the thick filament. This was in part because interactions of MyBP-C with the thick filament could adequately explain most (but not all) effects of MyBP-C on actomyosin interactions and in part because the specificity of actin binding was uncertain. However, numerous recent studies have now established that MyBP-C can indeed bind to actin through multiple binding sites, some of which are highly specific. Many of these interactions involve critical regulatory domains of MyBP-C that are also reported to interact with myosin. Here we review current evidence supporting MyBP-C interactions with actin and discuss these findings in terms of their ability to account for the functional effects of MyBP-C. We conclude that the influence of MyBP-C on muscle contraction can be explained equally well by interactions with actin as by interactions with myosin. However, because data showing that MyBP-C binds to either myosin or actin has come almost exclusively from in vitro biochemical studies, the challenge for future studies is to define which binding partner(s) MyBP-C interacts with in vivo.     

Thick filament assembly occurs after the formation of a cytoskeletal scaffold

The development of myofibrils involves the formation of contractile filaments and their assembly into the strikingly regular structure of the sarcomere. We analysed this assembly process in cultured human skeletal muscle cells and in rat neonatal cardiomyocytes by immunofluorescence microscopy using antibodies directed against cytoskeletal and contractile proteins. In particular, the question in which temporal order the respective proteins are integrated into developing sarcomeres was addressed. Although sarcomeric myosin heavy chain is expressed as one of the first myofibrillar proteins, its characteristic A band arrangement is reached at a very late stage. In contrast, titin, then myomesin and finally C-protein (MyBP-C) gradually form a regularly arranged scaffold on stress fiber-like structures (SFLS), on non-striated myofibrils (NSMF) and on nascent striated myofibrils (naSMF). Immediately subsequent to the completion of sarcomere cytoskeleton formation, the labeling pattern of myosin changes from the continuous staining of SFLS to the periodic staining characteristic for mature myofibrils. This series of events can be seen most clearly in the skeletal muscle cell cultures and – probably due to a faster developmental progression – less well in cardiomyocytes. We therefore conclude that the correct assembly of a cytoskeletal scaffold is a prerequisite for correct thick filament assembly and for the integration of the contractile apparatus into the myofibril.     

Single Particle Analysis: A new approach to solving the 3D structure of myosin filaments

Knowledge of the structure of muscle myosin filaments is essential for a proper understanding of sarcomere structure and how myosin heads interact with the actin filaments to produce force and movement. Two principal methods have been used to define the myosin head arrays in filaments in the relaxed state, namely modelling from low-angle X-ray diffraction data and image processing of electron micrographs of isolated filaments. Analysis of filament images by 3D helical reconstruction, which imposes total helical symmetry on the structure, is very effective in some cases, but it relies on the existence of very highly ordered preparations of straight filaments. Resolutions achieved to date are about 70 angstroms. Modelling of X-ray diffraction data recorded from whole relaxed fish or insect muscles has also been used as an independent method. Although the resolution of the diffraction data is often also about 70 angstroms, the effective resolution of the modelling is very much higher than this because additional very high resolution data (e.g. from protein crystallography) is included in the analysis. However, the X-ray diffraction method has to date provided only limited data on non-myosin thick filament proteins such as C-protein and titin and it cannot provide the polarity of the myosin head arrangement. Both the helical reconstruction and X-ray diffraction techniques have advantages and disadvantages, but their disadvantages are avoided in the new approach of single particle analysis of electron micrograph data. Even using the same micrographs as for helical reconstruction, the resolution can be extended by this method to about 50 angstroms or better. In addition, it is not necessary to assume that the myosin filaments are helical; a significant advantage in the case of vertebrate myosin filaments where there is a known crossbridge perturbation. Here we describe the principles of all these approaches, but particularly that of single particle analysis. We outline the application of single particle analysis to myosin filaments from vertebrate skeletal and insect flight (IFM) muscle myosin filaments.     

Effects of N-ethylmaleimide on the structure of skinned frog skeletal muscles

Summary The effects of N-ethylmaleimide (NEM) and other sulfhydryl modifiers on the structure of skinned frog skeletal muscles were studied using the X-ray diffraction technique. In sartorius muscle with full overlap between the thick and thin filaments, 0.1-1.0 mM NEM changed the intensity ratio of the (1,0) and (1,1) equatorial reflections from 4.35 to 0.72, and the (1,0) spacing of the hexagonal filament lattice from 40.4 to 41.4 nm. The axial X-ray diffraction pattern showed weak myosin layer-lines after the NEM treatment but enhancement of the actin layer-lines was not observed. In overstretched semitendinosus muscle, NEM did not affect the equatorial spacing but the myosin layer-lines were weakened. These results indicate that modification of myosin by NEM destroys the helical arrangement of myosin heads around the shaft of the thick filament and that when thin filaments are available, myosin heads move towards, and possibly bind to them. This binding is different from that in rigor since the ladder-like appearance of the higher actin layer-lines, which is typical of patterns from rigor muscles, was not observed. On removal of ATP after the NEM treatment, the diffraction pattern showed features characteristic of that from normal rigor muscles but no tension was produced. The pattern showed well-defined samplings on layer-lines in the small-angle region, indicating the presence of an extensive lattice order and exact axial alignment of the filaments. The first actin layer-line did not show samplings from the superlattice of the thick filaments, which are observed on the myosin layer-lines in patterns from resting muscles. This indicates that in NEM-treated rigor muscles the pattern of binding of myosin heads to the thin filaments is not influenced by the azimuthal orientation of the thick filament.     

A Spatially Explicit Nanomechanical Model of the Half-Sarcomere: Myofilament Compliance Affects Ca2+-Activation

The force exerted by skeletal muscle is modulated by compliance of tissues to which it is connected. Force of the muscle sarcomere is modulated by compliance of the myofilaments. We tested the hypothesis that myofilament compliance influences Ca2+ regulation of muscle by constructing a computational model of the muscle half sarcomere that includes compliance of the filaments as a variable. The biomechanical model consists of three half-filaments of myosin and 13 thin filaments. Initial spacing of motor domains of myosin on thick filaments and myosin-binding sites on thin filaments was taken to be that measured experimentally in unstrained filaments. Monte-Carlo simulations were used to determine transitions around a three-state cycle for each cross-bridge and between two-states for each thin filament regulatory unit. This multifilament model exhibited less "tuning" of maximum force than an earlier two-filament model. Significantly, both the apparent Ca(2+)-sensitivity and cooperativity of activation of steady-state isometric force were modulated by myofilament compliance. Activation-dependence of the kinetics of tension development was also modulated by filament compliance. Tuning in the full myofilament lattice appears to be more significant at submaximal levels of thin filament activation.     

Radial mass transfer of cross-bridges in a tetanized ferret heart muscle

An X-ray diffraction experiment on ferret heart muscle was made to examine the relationship between tension and mass transfer from the thick to the thin filament associated with the interaction of cross-bridges with actin. A ferret papillary muscle was electrically stimulated for 8 s in the presence of 5 microM ryanodine to give a tetanus. At different extracellular Ca2+ concentrations (2-20 mM), a linear relationship was found between the tension and the mass transfer. It was concluded that a change in tension caused by altering the extracellular Ca2+ concentration is due to a change in the number of myosin heads bound to actin. This is in contrast to the results obtained on skinned preparations, which showed a markedly nonlinear relationship between the number of heads in the vicinity of the thin filaments and force. It was found that the tension per myosin head is similar in twitch and tetanus of cardiac muscle. Also, a similar fraction of myosin heads is recruited in tetanus of cardiac and skeletal muscles.     

The structure of insect flight muscle in the presence of AMPPNP

Summary Glycerinated insect (Lethocerus) flight muscle in the presence of the non-hydrolysable ATP-analogue AMPPNP (1 mm at 4° C) has been prepared for electron microscopy using X-ray diffraction monitoring during fixation and embedding. Superior preservation of the original structure has been achieved through use of a fixative which included tannic acid and excess Mg²⁺. New features have been recognized in single filament layers (myac and actin) and 15 nm cross sections. As previously shown, some aspects of relaxed structure (14.5 nm shelves along thick filaments) and of rigor (38.7 nm angled bridges along thin filaments) are retained in a modified form. New observations include: (1) In 15 nm cross sections that show single 14.5 nm levels: (a) The flared X structure characteristic of rigor is replaced by a straight-X figure in which the crossbridge density is aligned along the myosin-actin plane, rather than skewed across it as in rigor, (b) In AMPPNP, each crossbridge appears to have a separate origin from the thick filament, rather than bifurcating two from one stem as in the flared X of rigor. The separation of crossbridge origins is also evidenced by the loss of ladder rungs in actin layers. (2) A halving (19.3 nm) of the 38.7 nm axial repeat along actin, rather than a thirding (12.9 nm) as in rigor, indicates redistribution of bridge attachments in cold AMPPNP. (3) In AMPPNP, the 14.5 nm shelves of density around the thick filament shaft are thicker but extend to smaller radius than similar shelves in ATP-relaxed muscle. This is shown by a lack of 14.5 nm periodicity and diffraction in actin layers of AMPPNP, in contrast to ATP-relaxed actin layers, in which the 14.5 nm period is present. Our results suggest that attached crossbridges are modified by AMPPNP and that ordered features of the analogue state are not accounted for solely by detached myosin heads or by a mixture of relaxed and rigor crossbridges. A two-domain model for the crossbridge is proposed. Domain 1 binds to the thin filament, and while bound, maintains a constant stereospecificity to actin at low resolution, independent of the type or presence of nucleotide at the myosin active site. Domain 2 is proximal to the thick filament and can exist in two characteristic states. In the absence of nucleotide (rigor), Domain 2 adopts a variable relationship to the thick filament, to accommodate the actin-bound end of the bridge. When ATP or AMPPNP are bound, in both attached and detached myosin heads, Domain 2 adopts a fixed disposition with respect to the thick filaments, its origin specified both longitudinally and azimuthally, while allowing a variable relationship to Domain 1 (nearest to actin). In AMPPNP, a variable relationship between the domains in attached crossbridges is required in order to connect the invariant 14.5 nm myosin origins to the 38.7nm actin target zones.     

Twitchin as a regulator of catch contraction in molluscan smooth muscle

Molluscan catch muscle can maintain tension for a long time with little energy consumption. This unique phenomenon is regulated by phosphorylation and dephosphorylation of twitchin, a member of the titin/connectin family. The catch state is induced by a decrease of intracellular Ca²⁺ after the active contraction and is terminated by the phosphorylation of twitchin by the cAMP-dependent protein kinase (PKA). Twitchin, from the well-known catch muscle, the anterior byssus retractor muscle (ABRM) of the mollusc Mytilus, incorporates three phosphates into two major sites D1 and D2, and some minor sites. Dephosphorylation is required for re-entering the catch state. Myosin, actin and twitchin are essential players in the mechanism responsible for catch during which force is maintained while myosin cross-bridge cycling is very slow. Dephosphorylation of twitchin allows it to bind to F-actin, whereas phosphorylation decreases the affinity of the two proteins. Twitchin has been also been shown to be a thick filament-binding protein. These findings raise the possibility that twitchin regulates the myosin cross-bridge cycle and force output by interacting with both actin and myosin resulting in a structure that connects thick and thin filaments in a phosphorylation-dependent manner.     

Vectorial phosphorylation of filamentous smooth muscle myosin by calmodulin and myosin light chain kinase complex

Vertebrate smooth muscle myosin extracted from myofibrils and isolated via filament assembly was co-purified with calmodulin (CaM) and myosin light chain kinase (MLCK) which are tightly associated with the filament architecture and, therefore, it may be considered as a native-like preparation. These endogenous contaminates also co-precipitated with a native-like actomyosin, for both cases, at levels sufficient to fully phosphorylate myosin within 10–20 s after addition of ATP and calcium, although their molar ratio to myosin was only about 1 to 100. Phosphorylation progress curves obtained from mixtures of the native-like, and CaM- and MLCK-free filaments indicated that the CaM/MLCK complex preferentially phosphorylated its parent filaments and, as result, the whole myosin present was not maximally phosphorylated. Solubilization of the filaments' mixtures at high ionic strength resulted in slower phosphorylation rates but with maximal phosphorylation levels being attainable. Similar observations were made on the filamentous myosin system reconstituted from the kinase- and CaM-free myosin with added purified MLCK and CaM as well as on the native-like myosin from which only one of these endogenous contaminates was removed by affinity chromatography. These data indicated that not only the MLCK but also CaM was necessary for the observed preferential phosphorylation kinetics. Thus, the native-like filamentous myosin appeared to be phosphorylated by some kind of vectorial mechanism. Similar experiments were carried out on the native-like actomyosin where these vectorial effects were even more pronounced.     

Distribution of developmental myosin isoforms in isolated A-segments

Summary Immunogold labelling was used to determine the distribution of myosin isoforms within the A-bands of developing chicken pectoralis muscles. Previous localization studies led to the suggestion that neonatal myosin is preferentially located in the centre of heterogeneous thick filaments that contain either embryonic or adult myosin in addition to neonatal myosin. To further explore the possibility that neonatal myosin may serve to nucleate thick filament assembly, a method was developed to isolate A-segments (arrays of myosin filaments) from myofibrils in the presence of MgATP. A-bands usually dissociate into thick and thin filaments in a relaxing buffer, but the inclusion of an antibody against M-line protein prevented separation of the thick filament array. Well-ordered A-segments, approximately 1.5 m in length, were prepared from muscles 12, 29, 40 days, and approximately 1 year after hatching. After reaction with monoclonal antibodies specific for neonatal and adult myosins, the A-segments were labelled with gold-conjugated secondary antibodies prior to negative staining. An antibody which cross-reacts with embryonic myosin was used to localize that epitope in A-bands of myofibrils from day 1 and day 3 posthatch muscles. At ages where expression of neonatal myosin was high, extensive gold labelling of A-segments was observed in the electron microscope. However, no preferential distribution of antibodies was observed at any age, independent of whether embryonic or adult myosin was coexpressed with the neonatal myosin, suggesting that neonatal myosin is not segregated to any particular region in the A-bands of developing muscles.     

Regulating the contraction of insect flight muscle

The rapid movement of the wings in small insects is powered by the indirect flight muscles. These muscles are capable of contracting at up to 1,000?Hz because they are activated mechanically by stretching. The mechanism is so efficient that it is also used in larger insects like the waterbug, Lethocerus. The oscillatory activity of the muscles occurs a low concentration of Ca(2+), which stays constant as the muscles contract and relax. Activation by stretch requires particular isoforms of tropomyosin and the troponin complex on the thin filament. We compare the tropomyosin and troponin of Lethocerus and Drosophila with that of vertebrates. The characteristics of the flight muscle regulatory proteins suggest ways in which stretch-activation works. There is evidence for bridges between troponin on thin filaments and myosin crossbridges on the thick filaments. Recent X-ray fibre diffraction results suggest that a pull on the bridges activates the thin filament by shifting tropomyosin from a blocking position on actin. The troponin bridges are likely to contain extended sequences of tropomyosin or troponin I (TnI). Flight muscle has two isoforms of TnC with different Ca(2+)-binding properties: F1 TnC is needed for stretch-activation and F2 TnC for isometric contractions. In this review, we describe the structural changes in both isoforms on binding Ca(2+) and TnI, and discuss how the steric model of muscle regulation can apply to insect flight muscle.     

Invertebrate muscles: thin and thick filament structure; molecular basis of contraction and its regulation, catch and asynchronous muscle

This is the second in a series of canonical reviews on invertebrate muscle. We cover here thin and thick filament structure, the molecular basis of force generation and its regulation, and two special properties of some invertebrate muscle, catch and asynchronous muscle. Invertebrate thin filaments resemble vertebrate thin filaments, although helix structure and tropomyosin arrangement show small differences. Invertebrate thick filaments, alternatively, are very different from vertebrate striated thick filaments and show great variation within invertebrates. Part of this diversity stems from variation in paramyosin content, which is greatly increased in very large diameter invertebrate thick filaments. Other of it arises from relatively small changes in filament backbone structure, which results in filaments with grossly similar myosin head placements (rotating crowns of heads every 14.5nm) but large changes in detail (distances between heads in azimuthal registration varying from three to thousands of crowns). The lever arm basis of force generation is common to both vertebrates and invertebrates, and in some invertebrates this process is understood on the near atomic level. Invertebrate actomyosin is both thin (tropomyosin:troponin) and thick (primarily via direct Ca(++) binding to myosin) filament regulated, and most invertebrate muscles are dually regulated. These mechanisms are well understood on the molecular level, but the behavioral utility of dual regulation is less so. The phosphorylation state of the thick filament associated giant protein, twitchin, has been recently shown to be the molecular basis of catch. The molecular basis of the stretch activation underlying asynchronous muscle activity, however, remains unresolved.     

Myotilin, a novel sarcomeric protein with two Ig-like domains, is encoded by a candidate gene for limb-girdle muscular dystrophy.

The striated muscle sarcomeres are highly organized structures composed of actin (thin) and myosin (thick) filaments that slide past each other during contraction. The integrity of sarcomeres is controlled by a set of structural proteins, among which are titin, a giant molecule that contains several immunoglobulin (Ig)-like domains and associates with thin and thick filaments, and [alpha]-actinin, an actin cross-linking protein. Mutations in several sarcomeric and sarcolemmal proteins have been shown to result in muscular dystrophy and cardiomyopathy. On the other hand, the disease genes underlying several disease forms remain to be identified. Here we describe a novel 57 kDa cytoskeletal protein, myotilin. Its N-terminal sequence is unique, but the C-terminal half contains two Ig-like domains homologous to titin. Myotilin is expressed in skeletal and cardiac muscle, it co-localizes with [alpha]-actinin in the sarcomeric I--bands and directly interacts with [alpha]-actinin. The human myotilin gene maps to chromosome 5q31 between markers AFM350yB1 and D5S500. The locus of a dominantly inherited limb-girdle muscular dystrophy (LGMD1A) resides in an overlapping narrow segment, and a new type of distal myopathy with vocal cord and pharyngeal weakness (VCPMD) has been mapped to the same locus. The muscle specificity and apparent role as a sarcomeric structural protein raise the possibility that defects in the myotilin gene may cause muscular dystrophy.     

A self-induced translation model of myosin head motion in contracting muscle I. Force-velocity relation and energy liberation

Summary In our previous model, it was assumed that the two heads of myosin act co-operatively in producing force for the sliding of actin filaments relative to myosin filaments. We eliminate the assumption of co-operativity in the present model, following the conclusion by Harada and co-workers that a co-operative interaction between the two heads of myosin is not essential in producing actin filament movement. We assume that (1) a myosin head activated by ATP hydrolysis binds to the thin filament at a definite angle and does not do the power stroke, i.e. does not change its orientation during attachment, (2) a potential of force acting on the myosin head is induced around the thin filament when an ATP-activated myosin head binds to an actin molecule in the thin filament, and (3) the potential remains for a while after detachment of the myosin head and statistically controls the direction of thermal motion of the myosin head, so that the myosin head translates toward the Z-line as a statistical average.We did calculations on these assumptions with a mean tension approximation and got the following results, (a) The calculated force-velocity relation in muscle contraction is in fairly good agreement with experimental observation, including the give phenomenon that lengthening velocity becomes very large for a force about twice the isometric tension. (b) The calculated rate of energy liberation during muscle contraction as a function of load on muscle is in good agreement with experimental results. (c) The calculated distance over which a myosin molecule moves along the thin filament during one ATP hydrolysis can be more than 60 nm under unloaded conditions.