Cross-striated Muscle Research Articles

Trichinella spiralis (Owen, 1835) Induces Increased Dystrophin Expression in Invaded Cross-striated Muscle.

Dystrophin and the dystrophin glycoprotein complex serve as a cytoskeletal integrator, critical for muscle membrane stability. The aim of the present study was to clarify the expression of dystrophin protein and mRNA in the skeletal muscle tissue during the muscle phase of trichinellosis in mice. Muscle tissue was collected from mice experimentally infected with Trichinella spiralis at days 0, 14 and 40 after infection. The expression of dystrophin in the muscle tissue was investigated by immunohistochemistry with antibodies against three different domains of the protein, and the expression levels of Dys mRNA by real-time PCR. The presence of dystrophin protein was increased in the de-differentiating cytoplasm at the early stage of muscle infection and was persisting also in the mature Nurse cell harbouring the parasite. It was accompanied by significantly elevated expression of Dys mRNA at days 14 and 40 after infection. Our findings indicate that dystrophin plays a role in regeneration of the muscle and in the Nurse cell formation and stability for security of the parasite survival.

Homozygous loss-of-function variants in FILIP1 cause autosomal recessive arthrogryposis multiplex congenita with microcephaly

Arthrogryposis multiplex congenita forms a broad group of clinically and etiologically heterogeneous disorders characterized by congenital joint contractures that involve at least two different parts of the body. Neurological and muscular disorders are commonly underlying arthrogryposis. Here, we report five affected individuals from three independent families sharing an overlapping phenotype with congenital contractures affecting shoulder, elbow, hand, hip, knee and foot as well as scoliosis, reduced palmar and plantar skin folds, microcephaly and facial dysmorphism. Using exome sequencing, we identified homozygous truncating variants in FILIP1 in all patients. FILIP1 is a regulator of filamin homeostasis required for the initiation of cortical cell migration in the developing neocortex and essential for the differentiation process of cross-striated muscle cells during myogenesis. In summary, our data indicate that bi-allelic truncating variants in FILIP1 are causative of a novel autosomal recessive disorder and expand the spectrum of genetic factors causative of arthrogryposis multiplex congenita.

A Histological Study on the Effect of Dextrose Prolotherapy on Skeletal Muscle Injury in Adult Male Albino Rats

Abstract Background Skeletal muscle injuries are one of the most common injuries occurring in sport medicine varies from 10% to 55% of all injuries. Phases of muscle injuries includes a series of complex stages including inflammation, regeneration and remodeling. Treatment of the injuries is based on conservative measures as rest, elevation, physical therapy and non steroidal anti-inflammatory medications. Many injection protocols have been proposed for the treatment of muscle lesions as corticosteroid injection. Recently prolotherapy appears to have a safety profile comparable with other injection procedures. Aim of the work The aim of the present work is to determine the efficacy of dextrose prolotherapy in treatment of skeletal muscle injury in adult male albino rats. Material and methods Sixty six adult male albino rats were used in the study. They were divided into control (group I) and experimental groups. Control group: rats were left without any intervention. The experimental group was group II that was divided into sham operated group in which skin incision was done in the left hindlimb without injury to gastrocnemius (group II`) and muscle injury group in which transverse cut injury across the midbelly of the gastrocnemius muscle of right hindlimb was done (group II``) and group III that was divided into lidocaine injected group in which skin incision in lefthindlimb was done without injury to gastrocnemius followed by injection of 0.3 ml 1% lidocaine was injected across the muscle (group III`) and muscle injury treated with dextrose prolotherapy group in which a transverse cut injury across the midbelly of gastrocnemius of right hindlimb was done followed by injection of 0.1 ml of dextrose prolotherapy of mixture of 0.1ml of 12.5% dextrose and 0.3 ml of 1% lidocaine (group III``). The animals were received 6 injections of lidocaine and dextrose prolotherapy at 5 days interval (starting from day 0 to day 25). In group II and III, muscle specimens were taken at 5,12 and 28 days and processed for light microscope Results Examination of Group II``A (5 days untreated group) showed intense infiltration of mononucleated inflammatory cells intermingling with dispersed myoblasts and macrophages. Group II`` B (12 days untreated group) showed regenerating myotubes intermingling with mononuclear inflammatory infiltrate and macrophages. Group II``C (28 days untreated group) showed some muscle fibers with peripherally elongated nuclei while others showed centrally vesicular ones. Examination of group III``A (5 day treated group with prolotherapy) showed longitudinal regenerating myofibers with multiple rows of internal vesiculated nuclei. Group III``B (12 days treated group) showed newly formed myofibers with incomplete striations together with well developed newly formed striated longitudinal muscle bundles with peripheral flattened nuclei, group III``C (28 days treated group) showed cross striated muscle fibers with the appearance of elongated vesicular nuclei. Conclusions dextrose prolotherapy was effective in soft tissue healing as it accelerated myoblast proliferation and differentiation

Myofibril and mitochondria morphogenesis are coordinated by a mechanical feedback mechanism in muscle

Complex animals build specialised muscles to match specific biomechanical and energetic needs. Hence, composition and architecture of sarcomeres and mitochondria are muscle type specific. However, mechanisms coordinating mitochondria with sarcomere morphogenesis are elusive. Here we use Drosophila muscles to demonstrate that myofibril and mitochondria morphogenesis are intimately linked. In flight muscles, the muscle selector spalt instructs mitochondria to intercalate between myofibrils, which in turn mechanically constrain mitochondria into elongated shapes. Conversely in cross-striated leg muscles, mitochondria networks surround myofibril bundles, contacting myofibrils only with thin extensions. To investigate the mechanism causing these differences, we manipulated mitochondrial dynamics and found that increased mitochondrial fusion during myofibril assembly prevents mitochondrial intercalation in flight muscles. Strikingly, this causes the expression of cross-striated muscle specific sarcomeric proteins. Consequently, flight muscle myofibrils convert towards a partially cross-striated architecture. Together, these data suggest a biomechanical feedback mechanism downstream of spalt synchronizing mitochondria with myofibril morphogenesis.

Exploring Obscurin and SPEG Kinase Biology.

Three members of the obscurin protein family that contain tandem kinase domains with important signaling functions for cardiac and striated muscles are the giant protein obscurin, its obscurin-associated kinase splice isoform, and the striated muscle enriched protein kinase (SPEG). While there is increasing evidence for the specific roles that each individual kinase domain plays in cross-striated muscles, their biology and regulation remains enigmatic. Our present study focuses on kinase domain 1 and the adjacent low sequence complexity inter-kinase domain linker in obscurin and SPEG. Using Phos-tag gels, we show that the linker in obscurin contains several phosphorylation sites, while the same region in SPEG remained unphosphorylated. Our homology modeling, mutational analysis and molecular docking demonstrate that kinase 1 in obscurin harbors all key amino acids important for its catalytic function and that actions of this domain result in autophosphorylation of the protein. Our bioinformatics analyses also assign a list of putative substrates for kinase domain 1 in obscurin and SPEG, based on the known and our newly proposed phosphorylation sites in muscle proteins, including obscurin itself.

Knockout of MYOM1 in human cardiomyocytes leads to myocardial atrophy via impairing calcium homeostasis.

Myomesin‐1 (encoded by MYOM1 gene) is expressed in almost all cross‐striated muscles, whose family (together with myomesin‐2 and myomesin‐3) helps to cross‐link adjacent myosin to form the M‐line in myofibrils. However, little is known about its biological function, causal relationship and mechanisms underlying the MYOM1‐related myopathies (especially in the heart). Regrettably, there is no MYMO1 knockout model for its study so far. A better and further understanding of MYOM1 biology is urgently needed. Here, we used CRISPR/Cas9 gene‐editing technology to establish an MYOM1 knockout human embryonic stem cell line (MYOM1−/− hESC), which was then differentiated into myomesin‐1 deficient cardiomyocytes (MYOM1−/− hESC‐CMs) in vitro. We found that myomesin‐1 plays an important role in sarcomere assembly, contractility regulation and cardiomyocytes development. Moreover, myomesin‐1‐deficient hESC‐CMs can recapitulate myocardial atrophy phenotype in vitro. Based on this model, not only the biological function of MYOM1, but also the aetiology, pathogenesis, and potential treatments of myocardial atrophy caused by myomesin‐1 deficiency can be studied.

A Histological Study on the Effect of Dextrose Prolotherapy on Skeletal Muscle Injury in Adult Male Albino Rats.

Background: Skeletal muscle injuries are one of the most common injuries in sport medicine. Many injection protocols have been proposed for the treatment as corticosteroid injection. Recently prolotherapy appears to have a safety profile comparable with other injection procedures. Aim of the work: was to determine the efficacy of dextrose prolotherapy in treatment of skeletal muscle injury in adult male albino rats.Material and methods: Sixty three adult male albino rats were used in the study. They were divided into control (group I). Group II was divided into sham operated group (group IIA) and muscle injury group (group IIB) and group III was divided into lidocaine injected group (group IIIA ) and muscle injury treated with dextrose prolotherapy group (group IIIB). Muscle specimens were taken at 5,12 and 28 days and processed for light microscopeResults: Examination of Group IIB1 (5 days) showed intense infiltration of mononucleated inflammatory cells intermingling with dispersed myoblasts and macrophages. Group IIB2 (12 days) showed regenerating myotubes intermingling with mononuclear inflammatory infiltrate and macrophages. Group IIB3 (28 days) showed some muscle fibers with peripherally elongated nuclei while others showed centrally vesicular ones. Examination of group IIIB1 (5 day treated) showed longitudinal regenerating myofibers with multiple rows of internal vesiculated nuclei. Group IIIB2 (12 days treated) showed newly formed myofibers with incomplete striations together with well developed newly formed striated longitudinal muscle bundles with peripheral flattened nuclei ,group IIIB3 (28 days treated) showed cross striated muscle fibers with the appearance of elongated vesicular nuclei.Conclusion: dextrose prolotherapy was effective in soft tissue healing as it accelerated myoblast proliferation and differentiation.

The Role of Cullin-RING Ligases in Striated Muscle Development, Function, and Disease.

The well-orchestrated turnover of proteins in cross-striated muscles is one of the fundamental processes required for muscle cell function and survival. Dysfunction of the intricate protein degradation machinery is often associated with development of cardiac and skeletal muscle myopathies. Most muscle proteins are degraded by the ubiquitin–proteasome system (UPS). The UPS involves a number of enzymes, including E3-ligases, which tightly control which protein substrates are marked for degradation by the proteasome. Recent data reveal that E3-ligases of the cullin family play more diverse and crucial roles in cross striated muscles than previously anticipated. This review highlights some of the findings on the multifaceted functions of cullin-RING E3-ligases, their substrate adapters, muscle protein substrates, and regulatory proteins, such as the Cop9 signalosome, for the development of cross striated muscles, and their roles in the etiology of myopathies.

Myofibril assembly and the roles of the ubiquitin proteasome system.

De novo assembly of myofibrils in vertebrate cross-striated muscles progresses in three distinct steps, first from a minisarcomeric alignment of several nonmuscle and muscle proteins in premyofibrils, followed by insertions of additional proteins and increased organization in nascent myofibrils, ending with mature contractile myofibrils. In a search for controls of the process of myofibril assembly, we discovered that the transition from nascent to mature myofibrils could be halted by inhibitors of three distinct functions of the ubiquitin proteasome system (UPS). First, inhibition of pathway to E3 Cullin ligases that ubiquitinate proteins led to an arrest of myofibrillogenesis at the nascent myofibril stage. Second, inhibition of p97 protein extractions of ubiquitinated proteins led to a similar arrest of myofibrillogenesis at the nascent myofibril stage. Third, inhibitors of proteolytic action by proteasomes also blocked nascent myofibrils from transitioning to mature myofibrils. In contrast, inhibitors of autophagy or lysosomes did not affect myofibrillogenesis. To probe for differences in the effects of UPS inhibitors during myofibrillogenesis, we analyzed by fluorescence recovery after photobleaching the exchange rates of two selected sarcomeric proteins (muscle myosin II heavy chains and light chains). In the presence of p97 and proteasomal inhibitors, the dynamics of each of these two myosin proteins decreased in the nascent myofibril stage, but were unaffected in the mature myofibril stage. The increased stability of myofibrils occurring in the transition from nascent to mature myofibril assembly indicates the importance of dynamics and selective destruction in the muscle myosin II proteins for the remodeling of nascent to mature myofibrils.

The origin of the heartbeat and theories of muscle contraction. Physiological concepts and conflicts in the 19th century

The origin of the incessant rhythmical heartbeat and the mechanism of muscle contraction have fascinated scientists over centuries. This short review outlines physiological explanations that were discussed in the 19th century starting with Albrecht von Haller (1708–1777), an 18th century physiologist who proposed that the heart has an intrinsic irritability. He argued that under normal conditions the inflow of blood stimulates the heart muscle to contract by mechanical touch and distension. Johannes Müller (1800–1858, physiologist in Bonn and Berlin) contended that the influence of the sympathetic nerve, specifically the activity of intracardiac ganglia, is the foremost cause of the heartbeat. Walter H. Gaskell and Theodor Engelmann (physiologists in Cambridge and Utrecht, respectively) independently criticized this neurogenic theory. They reported experimental evidence that supported the myogenic theory of the origin of the heartbeat, which has been accepted since about 1900. The concept of cardiac mechano-sensitivity, which can be traced back to A. von Haller, is currently resurging. Concerning mechanisms of contraction, Edward A. Schäfer (1850–1935), histologist and physiologist in Edinburgh, described differences between cardiac and skeletal muscle and coined the term sarcomere. Based on microscopic studies of cross-striated muscle, Schäfer outlined a detailed and plausible mechanism of muscle contraction in 1892. He put forward that during muscle shortening the “clear part of the muscle substance” (actin) might pass into longitudinal canals, which exist between the “sarcous elements” (myosin). His model foresaw fundamental elements of the sliding filament model, which was discovered by the Huxleys about 60 years later.

Phosphoproteomics identifies dual-site phosphorylation in an extended basophilic motif regulating FILIP1-mediated degradation of filamin-C

The PI3K/Akt pathway promotes skeletal muscle growth and myogenic differentiation. Although its importance in skeletal muscle biology is well documented, many of its substrates remain to be identified. We here studied PI3K/Akt signaling in contracting skeletal muscle cells by quantitative phosphoproteomics. We identified the extended basophilic phosphosite motif RxRxxp[S/T]xxp[S/T] in various proteins including filamin-C (FLNc). Importantly, this extended motif, located in a unique insert in Ig-like domain 20 of FLNc, is doubly phosphorylated. The protein kinases responsible for this dual-site phosphorylation are Akt and PKCα. Proximity proteomics and interaction analysis identified filamin A-interacting protein 1 (FILIP1) as direct FLNc binding partner. FILIP1 binding induces filamin degradation, thereby negatively regulating its function. Here, dual-site phosphorylation of FLNc not only reduces FILIP1 binding, providing a mechanism to shield FLNc from FILIP1-mediated degradation, but also enables fast dynamics of FLNc necessary for its function as signaling adaptor in cross-striated muscle cells.

Specialization for rapid excitation in fast squid tentacle muscle involves action potentials absent in slow arm muscle.

An important aspect of the performance of many fast muscle fiber types is rapid excitation. Previous research on the cross-striated muscle fibers responsible for the rapid tentacle strike in squid has revealed the specializations responsible for high shortening velocity, but little is known about excitation of these fibers. Conventional whole-cell patch recordings were made from tentacle fibers and the slower obliquely striated muscle fibers of the arms. The fast-contracting tentacle fibers show an approximately 10-fold greater sodium conductance than that of the arm fibers and, unlike the arm fibers, the tentacle muscle fibers produce action potentials. In situ hybridization using an antisense probe to the voltage-dependent sodium channel present in this squid genus shows prominent expression of sodium channel mRNA in tentacle fibers but undetectable expression in arm fibers. Production of action potentials by tentacle muscle fibers and their absence in arm fibers is likely responsible for the previously reported greater twitch-tetanus ratio in the tentacle versus the arm fibers. During the rapid tentacle strike, a few closely spaced action potentials would result in maximal activation of transverse tentacle muscle. Activation of the slower transverse muscle fibers in the arms would require summation of excitatory postsynaptic potentials over a longer time, allowing the precise modulation of force required for supporting slower movements of the arms.

Abstract 466: Myopathy Causing Bag3 P209L Protein Leads to Restrictive Cardiomyopathy Caused by Aggregate Formation and Sarcomere Disruption in Cardiomyocytes

The co-chaperone BAG3 (Bcl-2 associated athanogene 3) is strongly expressed in cross-striated muscles and plays a key role in the turnover of muscle-proteins as a member of the CASA (chaperone-assisted selected autophagy) complex. An amino acid exchange (P209L) in the human BAG3 gene, caused by a single base mutation, gives rise to a severe dominant childhood muscular dystrophy, restrictive cardiomyopathy, and respiratory insufficiency. To get deeper insights into the pathophysiological mechanisms of the disease, we generated a transgenic mouse model of the human mutation BAG3 P209L , in which a fusion protein consisting of the human BAG3 P209L and the green fluorescent protein eGFP can be conditionally overexpressed. Ubiquitous overexpression of BAG3 P209L -eGFP leads to a severe phenotype between the second and fourth week of life, including decreased body weight, skeletal muscle weakness, and heart failure. Echocardiography revealed that the BAG3 P209L -mice suffer from restrictive cardiomyopathy and Sirius-red-staining of heart tissue showed extensive fibrosis. In cardiomyocytes, isolated from hearts of transgenic mice overexpressing BAG3 wt -eGFP or BAG3 P209L -eGFP, BAG3 wt -eGFP stringently localizes to sarcomeres and intercalated discs, whereas cardiomyocytes from BAG3 P209L -eGFP mice displayed formation of BAG3 containing aggregates and disruption of sarcomeres in vivo . While BAG3 P209L -eGFP binding to á-Hsp70, Filamin C and á-HspB8 was unchanged it was less soluble than BAG3 and had a tendency to aggregate, thereby sequestering BAG3 and its clients. Depletion of the BAG3 pool leads to an impairment of CASA and accumulation of damaged proteins, causing sarcomere disintegration leading to restrictive cardiomyopathy.

Use of magnetic resonance imaging in the diagnosis of idiopathic inflammatory myopathies

Idiopathic inflammatory myopathies (IIMs) are a group of autoimmune diseases characterized by cross-striated muscle inflammation accompanied by muscle weakness. The rarity of these diseases and a large number of other diseases with similar clinical presentation, along with the absence of simple laboratory tests for confirming the diagnosis of IIMs can cause diagnostic difficulties. The latter may particularly frequently occur in the differentiation of polymyositis from sporadic inclusion body myositis, and late-onset limb-girdle muscular dystrophies. Magnetic resonance imaging of the thigh and leg muscles is of great importance for the differential diagnosis of myopathies, including autoimmune ones.

Results of the post-registration clinical study "PARUS" on efficiency and safety assessment of Mydocalm-Richter for local injection therapy of a myofascial trigger zone.

The "PARUS" program included investigation of the analgesic, muscle relaxant and sedative effects of Mydocalm-Richter which acts as central muscle relaxant in patients with myofascial pain syndrome, taking into account its registered indication for use - the hypertonus and cross-striated muscle spasm. Fifty patients with myofascial trigger points, the mean age of 41.67±11.86 years, have been enrolled in the study. All patients had undergone clinical examination that allowed the diagnosis of myofascial pain syndrome. The intensity of pain syndrome was evaluated using the pain visual analogue scales and McGill pain questionnaire. Visualization of area in spasm and evaluation of blood circulation was carried out using the ultrasound scan of target muscle. In order to objectively evaluate any conceivable hypotensive and sedative effects of Mydocalm-Richter we used the orthostatic test, Schulte's test for attention span and perfor-mance distribution and Munsterberg's test for attention discrimination and concentration. The analgesic and muscle relaxant effects of Mydocalm-Richter become apparent by day 3 post-injection, and the muscle relaxation effect is reaching its maximum on day 10 post-injection. Cardiovascular function following administration of Mydocalm-Richter was evaluated using the orthostatic test which revealed good orthostatic tolerance. Single injection of tolperisone hydrochloride possessing a central muscle relaxant activity has no sedative effect and does not influence patient response time. The ultrasound examination data demonstrated the improvement and in some cases restoration of blood circulation in the myofascial trigger points. Clinical study "PARUS" conducted in patients with myofascial pain has demonstrated a positive muscle relaxant and analgesic effect of Mydocalm-Richter that resulted in restoration of peripheral circulation in the myofascial trigger pointsconfirmed by ultrasound examination. An important benefit of this drug product is the absence of sedative effect and arterial hypotension.

Ordering of myosin II filaments driven by mechanical forces: experiments and theory

Myosin II filaments form ordered superstructures in both cross-striated muscle and non-muscle cells. In cross-striated muscle, myosin II (thick) filaments, actin (thin) filaments and elastic titin filaments comprise the stereotypical contractile units of muscles called sarcomeres. Linear chains of sarcomeres, called myofibrils, are aligned laterally in registry to form cross-striated muscle cells. The experimentally observed dependence of the registered organization of myofibrils on extracellular matrix elasticity has been proposed to arise from the interactions of sarcomeric contractile elements (considered as force dipoles) through the matrix. Non-muscle cells form small bipolar filaments built of less than 30 myosin II molecules. These filaments are associated in registry forming superstructures ('stacks') orthogonal to actin filament bundles. Formation of myosin II filament stacks requires the myosin II ATPase activity and function of the actin filament crosslinking, polymerizing and depolymerizing proteins. We propose that the myosin II filaments embedded into elastic, intervening actin network (IVN) function as force dipoles that interact attractively through the IVN. This is in analogy with the theoretical picture developed for myofibrils where the elastic medium is now the actin cytoskeleton itself. Myosin stack formation in non-muscle cells provides a novel mechanism for the self-organization of the actin cytoskeleton at the level of the entire cell.This article is part of the theme issue 'Self-organization in cell biology'.

DIAGNOSIS OF POISONING OF BEES WITH LEAD AND CADMIUM

Changes in the histogram of the midgut in bees exposed to lead and cadmium salts polluting their carbohydrate feed was studied. Normally , the mitgut is composed of the mucosa and muscular membranes separated with a thin layer of the basal membrane. The muscular membrane that forms the outer wall is a layer of cross-striated muscle symplasis intersecting in mutually perpendicular directions. On the mucous side, the muscular layer is delimited by a basal membrane and its outer side freely connects with internal environments of the abdominal region. Specific signs of lead poisoning manifest in the development of total and sub-total coagulative necrosis of the midgut mucous membrane. Its lesion in poisoning with cadmium is characterized by dystrophy expressed in cytoplasmic micro vesiculation which is followed by desquamation of the mucous membrane and destruction of glandular crypts.

Mast cells in damaged striated muscle of lymph heart in the adult frog: An ultrastructural, immunocytochemical, and auroradiographic study

We have studied mast-cell (MC) activation in the border area of mechanically damaged muscle in Rana temporaria lymph heart for 1 week after the surgery. Twenty-four hours after surgery, the cytoplasm of resident and circulating MCs contained degranulation channels discharging via compound exocytosis. Rare cases of single membrane-free expelled granules were revealed in the space limited with the plasma membrane of a resident MC and surrounding basal lamina. During the first week after the surgery the ultrastructural changes in many secretory granules of activated MCs concerned the granule swelling, reduced amount of the electron-dense component and pronounced loosing of their matrix. It is noteworthy that unaltered and altered granules are located close to empty degranulation channels frequently penetrated MC cytoplasm from the surface to centrally localized nucleus. Immunocytochemical MC labeling with antibodies to histamine revealed that gold particles were located under both altered and unaltered granules. In altered granules, gold particles were arranged above a preserved electron-dense part, whereas a loosened matrix, as a rule, was not labeled. Ultrastructural signs of secretory granule formation in the MC cytoplasm were disclosed to the end of the first week after surgery. Electron-microscopic autoradiography revealed that replicative and transcriptional activity of an MC in the border area was not prominent during all the periods after the surgery. Mitotic MCs were not observed. Occasionally, MCs dying via necrosis were encountered. These observations show that MCs stimulated by mechanical damage of the lymph heart secrete various preformed mediators, histamine in particular. The early response of MCs in the border area is evidence that activated MCs play a key role in the initiation of inflammation in the damaged cross-striated muscle of the lymph heart.

Myofibrillar Z-discs Are a Protein Phosphorylation Hot Spot with Protein Kinase C (PKCα) Modulating Protein Dynamics

The Z-disc is a protein-rich structure critically important for the development and integrity of myofibrils, which are the contractile organelles of cross-striated muscle cells. We here used mouse C2C12 myoblast, which were differentiated into myotubes, followed by electrical pulse stimulation (EPS) to generate contracting myotubes comprising mature Z-discs. Using a quantitative proteomics approach, we found significant changes in the relative abundance of 387 proteins in myoblasts versus differentiated myotubes, reflecting the drastic phenotypic conversion of these cells during myogenesis. Interestingly, EPS of differentiated myotubes to induce Z-disc assembly and maturation resulted in increased levels of proteins involved in ATP synthesis, presumably to fulfill the higher energy demand of contracting myotubes. Because an important role of the Z-disc for signal integration and transduction was recently suggested, its precise phosphorylation landscape further warranted in-depth analysis. We therefore established, by global phosphoproteomics of EPS-treated contracting myotubes, a comprehensive site-resolved protein phosphorylation map of the Z-disc and found that it is a phosphorylation hotspot in skeletal myocytes, underscoring its functions in signaling and disease-related processes. In an illustrative fashion, we analyzed the actin-binding multiadaptor protein filamin C (FLNc), which is essential for Z-disc assembly and maintenance, and found that PKCα phosphorylation at distinct serine residues in its hinge 2 region prevents its cleavage at an adjacent tyrosine residue by calpain 1. Fluorescence recovery after photobleaching experiments indicated that this phosphorylation modulates FLNc dynamics. Moreover, FLNc lacking the cleaved Ig-like domain 24 exhibited remarkably fast kinetics and exceedingly high mobility. Our data set provides research community resource for further identification of kinase-mediated changes in myofibrillar protein interactions, kinetics, and mobility that will greatly advance our understanding of Z-disc dynamics and signaling.

Mechanical tension and spontaneous muscle twitching precede the formation of cross-striated muscle in vivo.

Muscle forces are produced by repeated stereotypical actomyosin units called sarcomeres. Sarcomeres are chained into linear myofibrils spanning the entire muscle fiber. In mammalian body muscles, myofibrils are aligned laterally, resulting in their typical cross-striated morphology. Despite this detailed textbook knowledge about the adult muscle structure, it is still unclear how cross-striated myofibrils are built in vivo. Here, we investigate the morphogenesis of Drosophila abdominal muscles and establish them as an in vivo model for cross-striated muscle development. By performing live imaging, we find that long immature myofibrils lacking a periodic actomyosin pattern are built simultaneously in the entire muscle fiber and then align laterally to give mature cross-striated myofibrils. Interestingly, laser micro-lesion experiments demonstrate that mechanical tension precedes the formation of the immature myofibrils. Moreover, these immature myofibrils do generate spontaneous Ca2+-dependent contractions in vivo, which, when chemically blocked, result in cross-striation defects. Taken together, these results suggest a myofibrillogenesis model in which mechanical tension and spontaneous muscle twitching synchronize the simultaneous self-organization of different sarcomeric protein complexes to build highly regular cross-striated myofibrils spanning the length of large muscle fibers.