Myosin Thick Filaments Research Articles

Self-Organization in Reconstituted Actomyosin Bundles

Actomyosin bundles such as muscle myofibrils, stress fibers and cytokinetic rings are used by cells to exert force, contribute to the cell's structural integrity and accomplish morphological change. In muscle and some types of stress fibers actin, myosin and other components are organized into sarcomeric repeat units but in many other cases the structure is far more disordered. The mechanisms whereby such disordered architectures produce tension are not established. Here we used mathematical modeling to quantitatively understand the behavior of reconstituted in vitro actomyosin bundles consisting solely of F-actin and myosin thick filaments. In the presence of ADP, actin and myosin formed stable (>1 hour), non-tensile bundles ∼5-50 microns long anchored between polystyrene beads. The myosin is initially uniformly distributed along the bundles. Upon addition of ATP, the bundles contracted and became taut while the myosin reorganized into discrete clusters. We developed a mathematical model to account for this behavior. The random actin filament locations and polarities leads to a random net actin polarity at different locations along the bundle. We found the self-organization of myosins into clusters is driven by the tendency of myosin to migrate to zeros of the polarity profile. In agreement with experiment myosin clusters develop over ∼10 s by myosin translation. We calculate the distribution of myosin cluster separations and predict the mean cluster separation increases with actin filament length. Thus, a minimal bundle of actin and myosin alone has the inherent capacity to self-organize into a heterogeneous structure exhibiting morphological similarity to tension-producing cellular actomyosin structures such as stress fibers.

Cardiac Myosin Binding Protein-C (MYBP-C) Impedes Actin Filament Motility on Native Mouse Ventricular Thick Filaments Only Within the C-Zone

MyBP-C is a potent modulator of cardiac contractility and is localized to two distinct regions (C-zones) flanking the myosin thick filament (TF) bare zone. To determine whether MyBP-C modulates actomyosin motion generation over the entire length of the TF or only within the C-zones, we isolated native mouse ventricular TFs by mechanical and proteolytic dispersion. Negatively-stained EM images showed 1.6µm long TFs with defined central bare zones. TFs contained intact MyBP-C, confirmed by western blotting with domain specific antibodies. In a total internal reflectance microscopy based motility assay, TFs were imaged using fluorescent ATP and the velocity of fluorescent shards of actin filaments was measured over the entire length of TFs at 100 µM ATP and 20°C. Four distinct velocities were observed, presumably corresponding to motion toward and away from the bare zone, over regions with and without MyBP-C, the average velocity of which was similar to that generated by monomeric myosin in the motility assay (1.0±0.1µm/s). Limiting our analysis to bare zone directed motility, actin moved at 1.8±0.1µm/s starting from the TF end and slowed abruptly to 1.0±0.1µm/s at 285±8nm from the bare zone. In contrast, bare zone directed motility on TFs from MyBP-C knockout mice demonstrated only one velocity (1.7±0.1µm/s), equal to the faster velocity on wildtype (WT) TFs, suggesting the slow velocity on the WT TFs is due to MyBP-C. Similarly, only one bare zone directed velocity (1.9±0.1µm/s) was observed on WT TFs after N-terminal proteolytic truncation of the MyBP-C with calpain to within the MyBP-C motif (C1-C2 linker). We conclude that the N-terminal domains of MyBP-C directly modulate actomyosin motion generation only in a ∼285nm region of the thick filament, presumably the C-zone, where MyBP-C is localized.

Reconstitution of Actomyosin Bundles

Many vital processes among diverse cell types are dependent on proper actomyosin contraction. Despite muscle and non-muscle cells containing many different proteins believed to form the requisite structure necessary for contraction, the minimal requirements for effecting contractile motion and sustaining a tensile load are unknown. While myosin motors are known to be responsible for force generation, it is generally thought that long range force transmission requires accessory proteins to cross-link F-actin. We show that a minimal set of proteins, F-actin and smooth muscle myosin thick filaments, spontaneously assemble into contractile bundles that undergo dramatic reductions in contour length and generate contractile forces on the bundle end points. These contractile bundles are responsive to changes in boundary conditions, whereby dynamic remodeling seeks to minimize the distance and build tension between end points. The degree of contraction is sensitive to myosin/actin stoichiometries; at sufficiently low myosin concentration, contraction fails to commence. The rate of contractile is proportional to bundle length and, thus, these in vitro bundles show similar telescoping phenomenon observed in striated muscle. Contraction and further remodeling is abrogated at a critical tension, consistent with force-induced stalling of myosin motors within a bundle. Thus, we have found a two protein component system comprised of F-actin and thick filaments of smooth muscle myosin is sufficient to reconstitute a dynamic material that rapidly remodels through contraction when tension is low and forms a stable structure under high tension. This serves as a model to understand adaptive force sensing and transmission in the actomyosin cytoskeleton.

Myosin Binding Protein‐C: A Regulator of Actomyosin Interaction in Striated Muscle

Myosin-Binding protein-C (MyBP-C) is a family of accessory proteins of striated muscles that contributes to the assembly and stabilization of thick filaments, and regulates the formation of actomyosin cross-bridges, via direct interactions with both thick myosin and thin actin filaments. Three distinct MyBP-C isoforms have been characterized; cardiac, slow skeletal, and fast skeletal. Numerous mutations in the gene for cardiac MyBP-C (cMyBP-C) have been associated with familial hypertrophic cardiomyopathy (FHC) and have led to increased interest in the regulation and roles of the cardiac isoform. This review will summarize our current knowledge on MyBP-C and its role in modulating contractility, focusing on its interactions with both myosin and actin filaments in cardiac and skeletal muscles.

SM2+/- male mice are predisposed to develop urinary tract obstruction and hyper contractility of the bladder smooth muscle upon ageing

We previously showed that complete loss of smooth muscle myosin heavy chain isoform 2 (SM2) resulted in postnatal lethality, but in het mice a partial loss of SM2 (SM2(+/-)) was accompanied by down-regulation of SM1 with unaltered SM2:SM1 ratio. To determine whether a normal bladder function would be maintained throughout its lifespan, we aged WT and SM2(+/-) mice up to 18 months and analyzed a) SM2:SM1 ratio b) bladder smooth muscle structure and c) function in SM2(+/-) het mice. A notable finding was that ~50% of 15-18 months old male SM2(+/-) mice exhibited urinary retention in bladder with the distention of upper urethra. In SM2(+/-) mouse bladder with urinary retention, the SM2:SM1 ratio was decreased but not in SM2(+/-) mouse bladder that did not develop urinary retention. Interestingly in the distended bladder the expression levels of α-actin and tropomyosin remained unaltered despite a reduction in the number of myosin thick filaments. These distended bladders showed hypersensitivity to submaximal K(+) depolarization and M3-receptor stimulation, without a significant increase in myosin light chain phosphorylation. We therefore suggest that a partial loss of SM2 may predispose male mice to develop lower urinary tract obstruction during ageing. In addition our data suggest that bladder obstruction can cause a further reduction in SM2 expression and SM2:SM1 ratio, and a hyper-contractility of the bladder smooth muscle.

HIGHLIGHTS FROM THE LITERATURE

HIGHLIGHTS FROM THE LITERATURE

Knockdown and overexpression of Unc-45b result in defective myofibril organization in skeletal muscles of zebrafish embryos

BackgroundUnc-45 is a myosin chaperone and a Hsp90 co-chaperone that plays a key role in muscle development. Genetic and biochemical studies in C. elegans have demonstrated that Unc-45 facilitates the process of myosin folding and assembly in body wall muscles. Loss or overexpression of Unc-45 in C. elegans results in defective myofibril organization. In the zebrafish Danio rerio, unc-45b, a homolog of C. elegans unc-45, is expressed in both skeletal and cardiac muscles. Earlier studies indicate that mutation or knockdown of unc-45b expression in zebrafish results in a phenotype characterized by a loss of both thick and thin filament organization in skeletal and cardiac muscle. The effects of unc-45b knockdown on other sarcomeric structures and the phenotype of Unc-45b overexpression, however, are poorly understood in vertebrates.ResultsBoth knockdown and overexpression provide useful tools to study gene function during animal development. Using such methods, we characterized the role of Unc-45b in myofibril assembly of skeletal muscle in Danio rerio. We showed that, in addition to thick and thin filament defects, knockdown of unc-45b expression disrupted sarcomere organization in M-lines and Z-lines of skeletal muscles in zebrafish embryos. Western blotting analysis showed that myosin protein levels were significantly decreased in unc-45b knockdown embryos. Similarly, embryos overexpressing Unc-45b also exhibited severely disorganized myosin thick filaments. Disruption of thick filament organization by Unc-45b overexpression depends on the C-terminal UCS domain in Unc-45b required for interaction with myosin. Deletion of the C-terminal UCS domain abolished the disruptive activity of Unc-45b in myosin thick filament organization. In contrast, deletion of the N-terminal TPR domain required for binding with Hsp90α had no effect.ConclusionCollectively, these studies indicate that the expression levels of Unc-45b must be precisely regulated to ensure normal myofibril organization. Loss or overexpression of Unc-45b leads to defective myofibril organization.

Ultrastructural Study of Muscles Fibers in Tick Hyalomma (Hyalomma) anatolicum anatolicum (Ixodoidea: Ixodidae)

In the present study, ticks were obtained from a colony maintained at 28 degrees C and 75% relative humidity in at the Department of Biology, University College Umm Al-Qura University, Saudi Arabia and the Transmission Electron Microscope technique (TEM) was used to describes the ultrastructure and description of muscle of the of ixodid tick Hyalomma (Hyalomma) anatolicum anatolicum. The results showed that muscles of the unfed ticks Hyalomma (Hyalomma) anatolicum anatolicum in longitudinal sections are spindle-shaped to cylindrical muscle fibers. In the unfed nymph Hyalomma (Hyalomma) anatolicum anatolicum skeletal and visceral muscles are distinguished according to structure, function and position. These muscles include the capitulum, dorsoventral and leg oblique muscles. All muscle fibers are ensheathed (covered by sheath) in a sarcolemma. Their muscle fibers have striated pattern of successive sarcomeres whose thick myosin filaments are surrounded by orbitals of up to 12 thin actin filaments. The cytoplasm of the epidermal cell appears largely devoted with complicated microtubules present in parallel with long axis of adjacent muscle fibers. The cell membrane invaginates into tubular system extending deeply into the sarcoplasm and closely associated to cisternae of sarcoplasmic reticulum. The tubular system and sarcoplasmic reticulum forming two-membered (dyads) are considered to be the main route of calcium ions whose movement are synchronized with the motor impulse to control muscles contraction. In the sarcoplasm two types of muscle fibers are recognized according to thickness and density and mitochondrial size, distribution and population. Both skeletal and visceral muscles are invaginated by tracheoles and innervated by nerve axons containing synaptic vesicles. The actin and myosin filaments are slightly interrupted and the tubular system sarcoplasmic reticulum is well demonstrated.

Determinants of Myosin II Cortical Localization during Cytokinesis

Myosin II is an essential component of the contractile ring that divides the cell during cytokinesis. Previous work showed that regulatory light chain (RLC) phosphorylation is required for localization of myosin at the cellular equator. However, the molecular mechanisms that concentrate myosin at the site of furrow formation remain unclear. By analyzing the spatiotemporal dynamics of mutant myosin subunits in Drosophila S2 cells, we show that myosin accumulates at the equator through stabilization of interactions between the cortex and myosin filaments and that the motor domain is dispensable for localization. Filament stabilization is tightly controlled by RLC phosphorylation. However, we show that regulatory mechanisms other than RLC phosphorylation contribute to myosin accumulation at three different stages: (1) turnover of thick filaments throughout the cell cycle, (2) myosin heavy chain-based control of myosin assembly at the metaphase-anaphase transition, and (3) redistribution and/or activation of myosin binding sites at the equator during anaphase. Surprisingly, the third event can occur to a degree in a Rho-independent fashion, gathering preassembled filaments to the equatorial zone via cortical flow. We conclude that multiple regulatory pathways cooperate to control myosin localization during mitosis and cytokinesis to ensure that this essential biological process is as robust as possible.

Thermal activation energy for bidirectional movement of actin along bipolar tracks of myosin filaments

Previous in vitro motility assays using bipolar myosin thick filaments demonstrated that actin filaments were capable of moving in both directions along the myosin filament tracks. The movements; however, were slower in the direction leading away from the central bare zone than towards it. To understand the mechanism underlying these different direction-dependent motilities, we have examined the effects of temperature on the velocities of the bidirectional movements along reconstituted myosin filaments. Activation energies of the movements were determined by Arrhenius plots at high and low concentrations of ATP. As a result, the thermal activation energy of the movement away from the central bare zone was significantly higher than that of the movement toward the zone. Given that the backward movement away from the central bare zone would cause the myosin heads to be constrained and the stiffness of the cross-bridges to increase, these results suggest that elastic energy required for the cross-bridge transition is supplied by thermal fluctuations.

Identification of Novel Protein Kinase A Phosphorylation Sites in the M-domain of Human and Murine Cardiac Myosin Binding Protein-C Using Mass Spectrometry Analysis

Cardiac myosin binding protein-C (cMyBP-C) is a large multidomain accessory protein bound to myosin thick filaments in striated muscle sarcomeres. It plays an important role in the regulation of muscle contraction, and mutations in the gene encoding cMyBP-C are a common cause of familial hypertrophic cardiomyopathy, the leading cause of sudden cardiac death in young people. (1) The N-terminal domains including the C0, C1, cMyBP-C motif, and C2 domains play a crucial role in maintaining and modulating actomyosin interactions (keeping normal cardiac function) in a phosphorylation-dependent manner. The cMyBP-C motif or "M-domain" is a highly conserved linker domain in the N-terminus of cMyBP-C that contains three to five protein kinase A (PKA) phosphorylation sites, depending on species. For the human isoform, three PKA sites were previously identified (Ser(275), Ser(284), and Ser(304)), while three homologous sites exist in the murine isoform (Ser(273), Ser(282), and Ser(302)). The murine cMyBP-C isoform contains an additional conserved consensus site, Ser(307) that is not present in the human isoform. In this study, we investigated sites of PKA phosphorylation of murine and human cMyBP-C by treating the recombinant protein C0C2 ( approximately 50 KDa, which contains the N-terminal C0, C1, M, and C2 domains) and C1C2 (approximately 35 KDa, contains C1, M, and C2 domains) with PKA and assessing the phosphorylation states using SDS-PAGE with ProQ Diamond staining, and powerful hybrid mass spectrometric analyses. Both high-accuracy bottom-up and measurements of intact proteins mass spectrometric approaches were used to determine the phosphorylation states of C0C2 and C1C2 proteins with or without PKA treatment. Herein, we report for the first time that there are four PKA phosphorylation sites in both murine and human M-domains; both murine Ser(307) and a novel human Ser(311) can be phosphorylated in vitro by PKA. Future studies are needed to investigate the phosphorylation state of murine and human cMyBP-C in vivo.

Microscopic Measurement of Periodic Cross-Bridge Formation in Skeletal Myofibril

Electron microscopy and X-ray diffraction studies have been usually used for the examination of the interactions between actin and myosin, i.e., cross-bridge formation, in muscle fibers. These studies suggested the existence of “target zones [regions]” in actin (thin) filament, which are composed of three to four actin monomers that myosin heads in myosin thick filament can form cross-bridges. Direct evidence for the target zones, however, has been missing in myofibrils. Here we studied the interaction between a single actin filament and the thick filaments of rabbit skeletal myofibril under optical microscope. Single bead-tailed actin filament was manipulated by optical tweezers to make rigor cross-bridges on the outer surface of myofibril, and rupturing events were directly detected. We found the periodic cross-bridge formation, and frequently observed gaps, which are probably due to the incommensurate helical pitches between the thin and the thick filaments.

Automated Image Analysis of Electron Micrographs of Structurally Compromised Striated Muscle

Striated (skeletal and cardiac) muscle is a highly organized and conserved tissue with a molecular structure comprised of bundles of actin (thin filaments) and myosin (thick filaments). We have observed that skeletal muscle from two genetically modified murine models showing disrupted circadian rhythms (Bmal knockout and ClockΔ19), exhibit significant muscle weakness defined by a reduction in specific tension. Electron micrographs (EMs) of cross-sections from adult gastrocnemius in these mice reveal obvious divergences from the normal hexagonal arrangement of thin filaments around thick filaments.The goal of this project is to develop a tool for the high-throughput analysis of myofilament architecture. Image processing software written in MATLAB identifies myofilaments in EMs of muscle cross-sections as intensity peaks in the gray-scale image. Filaments are categorized as thick or thin depending on the cross-sectional area of the peaks after thresholding. Structural properties, such as the ratio of thin to thick filaments, the distance to closest neighbors, the angular distribution and the diameter of filaments will be determined for different muscle samples. This quantitative analysis should lead to improved understanding of structure-function relationships in striated muscle.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Conformation of A-Band Titin

The giant protein titin has important roles in the assembly, signalling and passive mechanical properties of muscle sarcomeres. Titin is formed by a single polypeptide with isoforms ranging between 3 and 4 MDa. This folds into ∼300 immunoglobulin (Ig) and fibronectin (Fn3) domains in a beads-on-a-string-like chain more than 1 μm long. The N-terminal half of the molecule forms an elastic connection between the end of the thick filament and the Z-line. The C-terminal half is bound to the thick (myosin) filament.

Molecular Structure of Sarcomere-to-Membrane Attachment at M-Lines inC. elegansMuscle

C. elegans is an excellent model for studying nonmuscle cell focal adhesions and the analogous muscle cell attachment structures. In the major striated muscle of this nematode, all of the M-lines and the Z-disk analogs (dense bodies) are attached to the muscle cell membrane and underlying extracellular matrix. Accumulating at these sites are many proteins associated with integrin. We have found that nematode M-lines contain a set of protein complexes that link integrin-associated proteins to myosin thick filaments. We have also obtained evidence for intriguing additional functions for these muscle cell attachment proteins.

Loss of Smyhc1 or Hsp90α1 Function Results in Different Effects on Myofibril Organization in Skeletal Muscles of Zebrafish Embryos

BackgroundMyofibrillogenesis requires the correct folding and assembly of sarcomeric proteins into highly organized sarcomeres. Heat shock protein 90α1 (Hsp90α1) has been implicated as a myosin chaperone that plays a key role in myofibrillogenesis. Knockdown or mutation of hsp90α1 resulted in complete disorganization of thick and thin filaments and M- and Z-line structures. It is not clear whether the disorganization of these sarcomeric structures is due to a direct effect from loss of Hsp90α1 function or indirectly through the disorganization of myosin thick filaments.Methodology/Principal FindingsIn this study, we carried out a loss-of-function analysis of myosin thick filaments via gene-specific knockdown or using a myosin ATPase inhibitor BTS (N-benzyl-p-toluene sulphonamide) in zebrafish embryos. We demonstrated that knockdown of myosin heavy chain 1 (myhc1) resulted in sarcomeric defects in the thick and thin filaments and defective alignment of Z-lines. Similarly, treating zebrafish embryos with BTS disrupted thick and thin filament organization, with little effect on the M- and Z-lines. In contrast, loss of Hsp90α1 function completely disrupted all sarcomeric structures including both thick and thin filaments as well as the M- and Z-lines.Conclusion/SignificanceTogether, these studies indicate that the hsp90α1 mutant phenotype is not simply due to disruption of myosin folding and assembly, suggesting that Hsp90α1 may play a role in the assembly and organization of other sarcomeric structures.

Binding of N-Terminus Fragments of Cardiac Myosin-Binding C-protein to Actin

Myosin-binding protein C (C-protein) is a sarcomeric protein regularly associated with thick filaments of vertebrate skeletal and heart muscle and is involved in the regulating of muscle contraction. C-protein belongs to the immunoglobulin- fibronectin superfamily of proteins and it consists of 11 tandemly arranged immunoglobulin-like domains. Four Ig-domains at the N-terminus of the cardiac isoform (C0-C1-m-C2 or C0C2) influence the actin -myosin S1 interaction whereas the C-terminal domains (C7-C10) play a structural role in the sarcomere, binding to myosin thick filaments. It has been shown that C0C2 fragment stabilizes F-actin, decorating the filaments in a highly regular arrangement.We used electron microscopy and single particle image analysis to reconstruct complexes of F-actin with cardiac C-protein fragments containing 4 Ig-domains (C0C2) and 2 Ig-domains (C0C1). Both fragments regularly decorated actin filaments and image analysis has revealed substantial rod-shaped density connecting adjacent monomers along the actin filament. The fragments induced dendritic growth of actin filaments in the first several minutes of co-polymerization followed by formation of regular sheets of filaments and ordered bundles. The results will be discussed in the terms of suggested involvement of C-protein in the modulation of contractile cycles of heart muscles.

Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation state

Cardiac myosin binding protein C (cMyBP-C), bound to the sarcomere's myosin thick filament, plays an important role in the regulation of muscle contraction. cMyBP-C is a large multidomain protein that interacts with myosin, titin, and possibly actin. Mutations in cMyBP-C are the most common known cause of heritable hypertrophic cardiomypathies. Phosphorylation of cMyBP-C plays an essential role in the normal cardiac function. cMyBP-C (142 kDa) has 81 serine and 73 threonine residues presenting a major challenge for unequivocal identification of specific phosphorylation sites. Top-down mass spectrometry, which directly analyzes intact proteins, is a powerful technique to universally observe and quantify protein posttranslational modifications without a priori knowledge. Here, we have extended top-down electron capture dissociation mass spectrometry to comprehensively characterize mouse cMyBP-C expressed in baculovirus. We have unambiguously identified all of the phosphorylation sites in the truncated (28-115 kDa) and full-length forms of cMyBP-C (142 kDa) and characterized the sequential phosphorylations, using a combination of top-down and middle-down (limited proteolysis) MS approach, which ensures full sequence coverage. Unit mass resolution and high mass accuracy (<5 ppm) have been achieved for a 115-kDa protein (the largest protein isotopically resolved to date). Remarkably, we discovered that truncations in recombinant proteins, even a seemingly minor one, can dramatically alter its phosphorylation state, which is significant because truncated recombinant proteins are routinely substituted for their full-length forms in crystal structure and functional studies. Our study provides direct evidence of alterations in the posttranslational state between the truncated and full-length recombinant proteins, which can lead to variations in structure and function.

CSN-5, a Component of the COP9 Signalosome Complex, Regulates the Levels of UNC-96 and UNC-98, Two Components of M-lines inCaenorhabditis elegansMuscle

In Caenorhabditis elegans two M-line proteins, UNC-98 and UNC-96, are involved in myofibril assembly and/or maintenance, especially myosin thick filaments. We found that CSN-5, a component of the COP9 signalosome complex, binds to UNC-98 and -96 using the yeast two-hybrid method. These interactions were confirmed by biochemical methods. The CSN-5 protein contains a Mov34 domain. Although one other COP9 signalosome component, CSN-6, also has a Mov34 domain, CSN-6 did not interact with UNC-98 or -96. Anti-CSN-5 antibody colocalized with paramyosin at A-bands in wild type and colocalized with abnormal accumulations of paramyosin found in unc-98, -96, and -15 (encodes paramyosin) mutants. Double knockdown of csn-5 and -6 could slightly suppress the unc-96 mutant phenotype. In the double knockdown of csn-5 and -6, the levels of UNC-98 protein were increased and the levels of UNC-96 protein levels were slightly reduced, suggesting that CSN-5 promotes the degradation of UNC-98 and that CSN-5 stabilizes UNC-96. In unc-15 and unc-96 mutants, CSN-5 protein was reduced, implying the existence of feed back regulation from myofibril proteins to CSN-5 protein levels. Taken together, we found that CSN-5 functions in muscle cells to regulate UNC-98 and -96, two M-line proteins.

The molecular effects of skeletal muscle myosin regulatory light chain phosphorylation

Phosphorylation of the myosin regulatory light chain (RLC) in skeletal muscle has been proposed to act as a molecular memory of recent activation by increasing the rate of force development, ATPase activity, and isometric force at submaximal activation in fibers. It has been proposed that these effects stem from phosphorylation-induced movement of myosin heads away from the thick filament backbone. In this study, we examined the molecular effects of skeletal muscle myosin RLC phosphorylation using in vitro motility assays. We showed that, independently of the thick filament backbone, the velocity of skeletal muscle myosin is decreased upon phosphorylation due to an increase in the myosin duty cycle. Furthermore, we did not observe a phosphorylation-dependent shift in calcium sensitivity in the absence of the myosin thick filament. These data suggest that phosphorylation-induced movement of myosin heads away from the thick filament backbone explains only part of the observed phosphorylation-induced changes in myosin mechanics. Last, we showed that the duty cycle of skeletal muscle myosin is strain dependent, consistent with the notion that strain slows the rate of ADP release in striated muscle.