Myofibrillogenesis in the developing chicken heart: role of actin isoforms and of the pointed end actin capping protein tropomodulin during thin filament assembly.

Recently, important differences between myofibrillogenesis in cultured cardiomyocytes vs. the three-dimensional setting in situ could be determined. We investigated thin filament assembly in situ by confocal microscopy of whole-mount preparations of immunostained embryonic chicken hearts. Of interest, a distinct localisation of different actin isoforms was observed in immature thin filaments. Cardiac alpha-actin is restricted to filaments with a length comparable to mature thin filaments as soon as the first contractions occur, while vascular alpha-actin makes up filaments that extend toward the M-band. The pointed-end actin filament capping protein tropomodulin can be found initially in close association with the plasma membrane, but attains its mature localisation pattern at the ends of the thin filaments only comparatively late during myofibrillogenesis. Thus tropomodulin acts as a length stabilising element of actin filaments also in developing cardiomyocytes in situ, but plays an additional role together with membrane-associated actin filaments in the earliest steps of myofibril assembly.     

Tropomyosin is required for cardiac morphogenesis,myofibril assembly,and formation of adherens junctions in the developing mouse embryo

BACKGROUND: We explored a function for tropomyosin (TM) in mammalian myofibril assembly and cardiac development by analyzing a deletion in the mouse TPM1 gene targeting αTM1, the major striated muscle TM isoform. RESULTS: Mice lacking αTM1 are embryonic lethal at E9.5 with enlarged, misshapen, and non‐beating hearts characterized by an abnormally thin myocardium and reduced trabeculae. αTM1‐deficient cardiomyocytes do not assemble striated myofibrils, instead displaying aberrant non‐striated F‐actin fibrils with α‐actinin puncta dispersed irregularly along their lengths. αTM1's binding partner, tropomodulin1 (Tmod1), is also disorganized, and both myomesin‐containing thick filaments as well as titin Z1Z2 fail to assemble in a striated pattern. Adherens junctions are reduced in size in αTM1‐deficient cardiomyocytes, α‐actinin/F‐actin adherens belts fail to assemble at apical cell–cell contacts, and cell contours are highly irregular, resulting in abnormal cell shapes and a highly folded cardiac surface. In addition, Tmod1‐deficient cardiomyocytes exhibit failure of α‐actinin/F‐actin adherens belt assembly. CONCLUSIONS: Absence of αTM1 resulting in unstable F‐actin may preclude sarcomere formation and/or lead to degeneration of partially assembled sarcomeres due to unregulated actomyosin interactions. Our data also identify a novel αTM1/Tmod1‐based pathway stabilizing F‐actin at cell–cell junctions, which may be required for maintenance of cell shapes during embryonic cardiac morphogenesis. Developmental Dynamics 243:800–817, 2014. © 2014 Wiley Periodicals, Inc.     

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.     

Calcium transients regulate patterned actin assembly during myofibrillogenesis.

The highly ordered arrangement of sarcomeric myosin during striated muscle development requires spontaneous calcium (Ca(2+)) transients. Here, we show that blocking transients also compromises patterned assembly of actin thin filaments, titin, and capZ. Because a conserved temporal assembly pattern has been described for these proteins, selective inhibitors of either thick or thin filament formation were used to determine their relative temporal interdependencies. For example, inhibition of myosin light chain kinase (MLCK) by application of a specific inhibitory peptide or phorbol myistate acetate (PMA) disrupts myosin assembly without significantly affecting formation of actin bands. The MLCK inhibitor ML-7, however, disrupted actin as well as myosin. Surprisingly, agents that interfere with actin dynamics, such as cytochalasin D, produced only minor organizational disruptions in actin, capZ, and titin staining. However, cytochalasin D and other actin disrupting compounds significantly perturbed myosin organization. The results indicate that (1) Ca(2+) transients regulate one or more of the earliest steps in sarcomere formation, (2) mature actin filaments can assemble independently of myosin band formation, and (3) myosin thick filament assembly is extremely sensitive to disruption of either the actin or titin filament systems.     

Interaction between titin and thin filaments in intact cardiac muscle

A freeze break technique and immunoelectronmicroscopy were used to study the elastic properties of cardiactitin filaments. Small bundles consisting of a few fibres fromfreshly prepared dog papillary muscle were quickly frozen andbroken under liquid nitrogen to fracture sarcomeres in planesperpendicular to the filament axes. Breaks occurred at each ofseveral regions along the sarcomeres. The still-frozen specimenswere thawed during fixation to allow elastic filaments toretract. The broken muscle segments were then treated withmonoclonal titin antibody 9D10 which labelled a unique epitope inthe I-band. In sarcomeres broken at the A-I junction, the titinfilaments reacted toward the Z-line, independently of the thinfilaments. The retracted epitopes did not reach the Z-line;retraction stopped at the N1-line level. In sarcomeres brokennear the Z-line, the titin filaments retracted in the oppositedirection, to the tip of the thick filaments. When the breakoccurred in the A-band, by contrast, the titin-epitope positionwas unaffected. On the basis of these results, and despite thereported interaction of titin and actin in vitro, it appears thatcardiac titin molecules form elastic filaments that arefunctionally independent of the thin filaments. Near the Z-line,however, the titin filaments seem to associate firmly with thethin filaments     

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.     

Immunocytochemical studies using a monoclonal antibody to bovine cardiac titin on intact and extracted myofibrils

Summary A monoclonal antibody specific to bovine cardiac titin has been identified. The antibody recognizes a common antigenic site in striated muscles of several species. In relaxed myofibrils, specific staining at the A–I junction resulted in a doublet of fluorescent bands within a sarcomere. The distance between the doublets in successive sarcomeres varied according to the degree of myofibrillar contraction. Staining on formamide-extracted myofibrils has confirmed that this epitope is located near the outer edges of isolated A bands. Selective extraction of myofibrillar proteins resulted in different staining patterns. Disrupting the structural integrity of the M-line or the A-band centre caused a significant amount of titin to translocate toward the Z-line region. In contrast, shortening of the A-band by removal of myosin from the ends of the thick filaments resulted in anti-titin staining moving closer to the M-line region. Several conclusions can be drawn from this study: (a) two aligned groups of titin molecules are placed symmetrically to the M-line in a sarcomere; (b) titin may attach directly or via intermediary protein(s) to sites near the M-line and Z-line such that the protein is under tension and (c) removal of proteins from either region results in titin staining in the opposite region. However, the edges of the A-band give some hindrance to collaspe of the titin toward the M-line.     

Fish muscle cytoskeleton integrity is not dependent on intact thin filaments

Striated musclecytoskeleton was studied by ultrastructure and electrophoresis.Treatment of sea bass white muscle myofibrils and glycerinatedfibres with calpain caused disruption of costameres, intermediatefilaments, and Z-line, without altering sarcomeres. V8 proteasealso caused loss of costameres and Z-line, and disruptedsarcomeres without affecting the intermediate filaments.Recombinant lipase caused loss of Z-lines and also sarcolemmadetachment, without changing sarcomeres or intermediatefilaments. DNase-1 removed thin filaments and partially removedZ-lines while leaving intact the sarcolemma attachments andintermediate filaments. Calpain, V8 protease, lipase and DNase-1treatments induced extensive loss of -actinin from the Z-line, which could be related to titin cleavage (calpain, V8),phosphoinositide hydrolysis (lipase), and actin depolymerisation(DNase-1). These results show that the cytoskeletal componentsare independent of intact thin filaments     

How to build a myofibril

Building a myofibril from its component proteins requires the interactions of many different proteins in a process whose details are not understood. Several models have been proposed to provide a framework for understanding the increasing data on new myofibrillar proteins and their localizations during muscle development. In this article we discuss four current models that seek to explain how the assembly occurs in vertebrate cross-striated muscles. The models hypothesize: (a) stress fiber-like structures as templates for the assembly of myofibrils, (b) assembly in which the actin filaments and Z-bands form subunits independently from A-band subunits, with the two subsequently joined together to form a myofibril, (c) premyofibrils as precursors of myofibrils, or (d) assembly occurring without any intermediary structures. The premyofibril model, proposed by the authors, is discussed in more detail as it could explain myofibrillogenesis under a variety of different conditions: in ovo, in explants, and in tissue culture studies on cardiac and skeletal muscles.In memoriam: This paper is dedicated to the memory of Professor Koscak Maruyama, a noted contributor in the field of muscle biochemistry.     

Myotilin,the limb-girdle muscular dystrophy 1A (LGMD1A) protein,cross-links actin filaments and controls sarcomere assembly

The assembly and maintenance of the muscle sarcomere requires a complex interplay of actin- and myosin-associated proteins. Myotilin is a thin filament-associated Z-disc protein that consists of two Ig-domains flanked by a unique serine-rich amino-terminus and a short carboxy-terminal tail. It binds to alpha-actinin and filamin c and is mutated in limb girdle muscular dystrophy 1A (LGMD1A). Here we show that myotilin also directly binds F-actin, efficiently cross-links actin filaments alone or in concert with alpha-actinin and prevents filament disassembly induced by Latrunculin A. Myotilin forms dimers via its carboxy-terminal half, which may be necessary for the actin-bundling activity. Overexpression of full-length myotilin but not the carboxy-terminal half induces formation of thick actin cables in non-muscle cells devoid of endogenous myotilin. The expression of myotilin in muscle cells is tightly regulated to the later stages of in vitro myofibrillogenesis, when preassembled myofibrils begin to align. Expression of either amino- or carboxy-terminally truncated myotilin fragments but not wild-type myotilin in differentiating myocytes leads to myofibril disarray. The disease association and functional characteristics indicate an indispensable role for myotilin in stabilization and anchorage of thin filaments, which may be a prerequisite for correct Z-disc organization.     

Stabilization and remodeling of the membrane skeleton during lens fiber cell differentiation and maturation.

Actin filaments are integral components of the plasma membrane-associated cytoskeleton (membrane skeleton) and are believed to play important roles in the determination of cell polarity, shape, and membrane mechanical properties, however the roles of actin regulatory proteins in controlling the assembly, stability, and organization of actin filaments in the membrane skeleton are not well understood. Tropomodulin is a tropomyosin and actin-binding protein that stabilizes tropomyosin-actin filaments by capping their pointed ends and is associated with the spectrin-actin membrane skeleton in erythrocytes, skeletal muscle cells, and lens fiber cells, a specialized epithelial cell type. In this study, we have investigated the role of tropomodulin and other membrane skeleton components in lens fiber cell differentiation and maturation. Our results demonstrate that tropomodulin is expressed concomitantly with lens fiber cell differentiation and assembles onto the plasma membrane only after fiber cells have begun to elongate and form apical-apical contacts with the undifferentiated epithelium. In contrast, other membrane skeleton components, spectrin, actin, and tropomyosin, are constitutively expressed and assembled on the plasma membranes of both undifferentiated and differentiated fiber cells. Tropomodulin, but not other membrane skeleton components, is also enriched at a novel structure at the apical and basal ends of newly elongated fiber cells at the fiber cell-epithelium and fiber cell-capsule interface, respectively. Once assembled, tropomodulin and its binding partners, tropomyosin and actin, remain membrane-associated and are not proteolyzed during fiber cell maturation and aging, despite proteolysis of alpha-spectrin and other cytoskeletal filament systems such as microtubules and intermediate filaments. We propose that actin filament stabilization by tropomodulin, coupled with partial proteolysis of other cytoskeletal components, represents a programmed remodeling of the lens membrane skeleton that may be essential to maintain plasma membrane integrity and transparency of the extremely elongated, long-lived cells of the lens. The unique localization of tropomodulin at fiber cell tips further suggests a new role for tropomodulin at cell-cell and cell-substratum contacts; this may be important for cell migration and/or adhesion during differentiation and morphogenesis.     

Cardiac titin: molecular basis of elasticity and cellular contribution to elastic and viscous stiffness components in myocardium

Myocardium resists the inflow of blood during diastole through stretch-dependent generation of passive tension. Earlier we proposed that this tension is mainly due to collagen stiffness at degrees of stretch corresponding to sarcomere lengths (SLS) ≥2.2 μm, but at shorter lengths, is principally determined by the giant sarcomere protein titin. Myocardial passive force consists of stretch-velocity-sensitive (viscous/viscoelastic) and velocity-insensitive (elastic) components; these force components are seen also in isolated cardiac myofibrils or skinned cells devoid of collagen. Here we examine the cellular/myofibrillar origins of passive force and describe the contribution of titin, or interactions involving titin, to individual passive-force components. We construct force–extension relationships for the four distinct elastic regions of cardiac titin, using results of in situ titin segment-extension studies and force measurements on isolated cardiac myofibrils. Then, we compare these relationships with those calculated for each region with the wormlike-chain (WLC) model of entropic polymer elasticity. Parameters used in the WLC calculations were determined experimentally by single-molecule atomic force-microscopy measurements on engineered titin domains. The WLC modelling faithfully predicts the steady-state-force vs. extension behavior of all cardiac-titin segments over much of the physiological SL range. Thus, the elastic-force component of cardiac myofibrils can be described in terms of the entropic-spring properties of titin segments. In contrast, entropic elasticity cannot account for the passive-force decay of cardiac myofibrils following quick stretch (stress relaxation). Instead, slower (viscoelastic) components of stress relaxation could be simulated by using a Monte-Carlo approach, in which unfolding of a few immunoglobulin domains per titin molecule explains the force decay. Fast components of stress relaxation (viscous drag) result mainly from interaction between actin and titin filaments; actin extraction of cardiac sarcomeres by gelsolin immediately suppressed the quickly decaying force transients. The combined results reveal the sources of velocity sensitive and insensitive force components of cardiomyofibrils stretched in diastole. This revised version was published online in July 2006 with corrections to the Cover Date.     

Obscurin: a multitasking muscle giant

Obscurin (~800 kDa) is the third member of a family of giant proteins expressed in vertebrate striated muscle, along with titin (3–3.7 MDa) and nebulin (~800 kDa). Like its predecessors, it is a multidomain protein composed of tandem adhesion modules and signaling domains. Unlike titin and nebulin, which are integral components of sarcomeres, obscurin is concentrated at the peripheries of Z-disks and M-lines, where it is appropriately positioned to communicate with the surrounding myoplasm. This unique distribution allows obscurin to bind small ankyrin 1, an integral component of the sarcoplasmic reticulum (SR) membrane. Obscurin also associates with the contractile apparatus through its binding to titin, sarcomeric myosin and perhaps other proteins of the contractile apparatus. Overexpression of the COOH-terminus of obscurin in primary myotubes has a dramatic and specific effect on the organization of sarcomeric myosin, indicating a role in the organization and regular assembly of A-bands. Given its ability to associate tightly, selectively and periodically with the periphery of the myofibril, its high affinity for an integral membrane protein of the SR and its close association with thick filaments, we speculate that obscurin is ideally suited to play key roles in modulating the organization and assembly of both the myofibril and the SR.     

Regulation of Structure and Function of Sarcomeric Actin Filaments in Striated Muscle of the Nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans has been used as a valuable system to study structure and function of striated muscle. The body wall muscle of C. elegans is obliquely striated muscle with highly organized sarcomeric assembly of actin, myosin, and other accessory proteins. Genetic and molecular biological studies in C. elegans have identified a number of genes encoding structural and regulatory components for the muscle contractile apparatuses, and many of them have counterparts in mammalian cardiac and skeletal muscles or striated muscles in other invertebrates. Applicability of genetics, cell biology, and biochemistry has made C. elegans an excellent system to study mechanisms of muscle contractility and assembly and maintenance of myofibrils. This review focuses on the regulatory mechanisms of structure and function of actin filaments in the C. elegans body wall muscle. Sarcomeric actin filaments in C. elegans muscle are associated with the troponin–tropomyosin system that regulates the actin–myosin interaction. Proteins that bind to the side and ends of actin filaments support ordered assembly of thin filaments. Furthermore, regulators of actin dynamics play important roles in initial assembly, growth, and maintenance of sarcomeres. The knowledge acquired in C. elegans can serve as bases to understand the basic mechanisms of muscle structure and function. Anat Rec, 297:1548–1559, 2014. © 2014 Wiley Periodicals, Inc.     

Quantal length changes in single contracting sarcomeres

The time course of shortening was investigated in the single sarcomere, the smallest contractile unit that retains natural structure. We projected the striation patterns of single bumblebee flight-muscle myofibrils onto a linear photodiode array, which was scanned periodically to produce repetitive traces of intensity vs. position along the array. Sarcomere length was taken as the span between adjacent A-band or Z-line centroids. When myofibrils were ramp-released by a motor, individual sarcomeres shortened in steps punctuated by pauses. The single sarcomere-shortening trace was consistently stepwise both in activated and relaxed specimens. Although step size was variable, the size distribution showed a signature-like feature: the histogram comprised distinct peaks that were spaced quasi-regularly. In the activated myofibrils the interpeak separation corresponded to 2.71 nm per half-sarcomere. This value is equal to the linear advance of actin subunits along the thin filament. Thus, actin filaments translate over thick filaments by steps that may be integer multiples of the actin-subunit spacing.     

Ultrastructural changes in myocardial cells of rats fed a low protein diet

 Ultrastructural changes in the ventricular myocardial cells in rats fed a low protein diet were examined by electron microscopy. The most striking changes were observed in the I-band region of the sarcomeres, which occurred very occasionally in myofibrils. In the sarcomere affected the I-band region was often fractured and/or disintegrated on one side, leaving an extended space, while the opposing I-band region disappeared along with dislocation of the intact A-band toward the adjacent Z-line. This dislocation was presumably attributed to the elasticity of titins connecting between the end of thick filaments and the Z-line. Fractured I-band regions were often accompanied by the dilated sarcoplasmic reticulum in the close vicinity of them. In some myofibrils the streaming and/or disruption of the Z-line were occasionally observed where disarrangement of thick and thin myofilaments were usually present. The study suggests that the fracture of the I-band region, consisting of actin and titin filaments, and the streaming of the Z-line of myofibrils are due to a proteolytic action of calpain and/or cathepsin L, which are activated by leaked Ca²⁺ ion and/or by modification of internal circumstances of the cytoplasm induced by a low protein diet, thus resulting in a low cardiac output. Received: 22 April 1998 / Accepted: 7 September 1998     

The ultrastructure of skeletal and smooth muscle in experimental protein malnutrition in rats fed a low protein diet

Light microscopy of the pectoralis muscle of rats on a low protein diet did not show such morphological alterations as atrophy, degeneration, or sarcoplasmic edema, but electron microscopy occasionally demonstrated ultrastructural changes only in the sarcomeres of myofibrils. In the affected sarcomeres, the Z-line was disrupted and often showed a jagged structure. The Z-substance with electron opacity was frequently present flowing along the long axis of myofibrils, here referred to as the streaming of Z-lines. In addition, regular striations formed by the reciprocal arrangement of thick and thin filaments disappeared from the affected sarcomeres, though these filaments were still discernible. Two or more consecutive sarcomeres in a single myofibril were occasionally involved in these changes. A further two or more neighboring sarcomeres at the same level of myofibrils were affected transversely by these structural alterations. On the other hand, the ultrastructure of the intestinal smooth muscle was not affected by protein deficiency. The study suggests that the ultrastructural damage induced by a low protein diet is attributed to the activation of endogenous protease by the excess leaking of Ca2+ into the cytosol as a result of lipid peroxidation of cell membrane by raised free radicals, owing to the depletion of glutathione production by protein deficiency. It also suggests that the smooth muscle cells differ in their susceptibility to protein deficiency from the skeletal muscle cells.     

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.     

Residual force enhancement in skeletal muscles: one sarcomere after the other

The force-length relation is one of the most prominent features of striated muscles, and predicts that the force produced by a fully activated muscle is proportional to the overlap between myosin and actin filaments within sarcomeres. However, there are situations in which the force-length relation deviates from predictions based purely on filament overlap. Notably, stretch of activated skeletal muscles induces a long-lasting increase in force, which is larger than the force produced during isometric contractions at a similar length. The mechanism behind this residual force enhancement and deviations from the original force-length relation are unknown, generating heated debate in the literature. We performed a series of experiments with short segments of myofibrils and isolated sarcomeres to investigating the mechanisms of the residual force enhancement and the force length-relation. In this paper, evidence will be presented showing that force enhancement is caused by: (i) half-sarcomere non-uniformities, and (ii) a sarcomeric component, which may be associated with Ca(2+)-induced stiffness of titin molecules. These mechanisms have large implications for understanding the basic mechanisms of muscle contraction.     

Obscurin is required for the lateral alignment of striated myofibrils in zebrafish.

Obscurin/obscurin-MLCK is a giant sarcomere-associated protein with multiple isoforms whose interactions with titin and small ankyrin-1 suggest that it has an important role in myofibril assembly, structural support, and the sarcomeric alignment of the sarcoplasmic reticulum. In this study, we characterized the zebrafish orthologue of obscurin and examined its role in striated myofibril assembly. Zebrafish obscurin was expressed in the somites and central nervous system by 24 hours post-fertilization (hpf) and in the heart by 48 hpf. Depletion of obscurin using two independent morpholino antisense oligonucleotides resulted in diminished numbers and marked disarray of skeletal myofibrils, impaired lateral alignment of adjacent myofibrils, disorganization of the sarcoplasmic reticulum, somite segmentation defects, and abnormalities of cardiac structure and function. This is the first demonstration that obscurin is required for vertebrate cardiac and skeletal muscle development. The diminished capacity to generate and organize new myofibrils in response to obscurin depletion suggests that it may have a vital role in the causation of or adaptation to cardiac and skeletal myopathies.