Cytoskeletal protein kinases: titin and its relations in mechanosensing

Titin, the giant elastic ruler protein of striated muscle sarcomeres, contains a catalytic kinase domain related to a family of intrasterically regulated protein kinases. The most extensively studied member of this branch of the human kinome is the Ca2+–calmodulin (CaM)-regulated myosin light-chain kinases (MLCK). However, not all kinases of the MLCK branch are functional MLCKs, and about half lack a CaM binding site in their C-terminal autoinhibitory tail (AI). A unifying feature is their association with the cytoskeleton, mostly via actin and myosin filaments. Titin kinase, similar to its invertebrate analogue twitchin kinase and likely other “MLCKs”, is not Ca2+–calmodulin-activated. Recently, local protein unfolding of the C-terminal AI has emerged as a common mechanism in the activation of CaM kinases. Single-molecule data suggested that opening of the TK active site could also be achieved by mechanical unfolding of the AI. Mechanical modulation of catalytic activity might thus allow cytoskeletal signalling proteins to act as mechanosensors, creating feedback mechanisms between cytoskeletal tension and tension generation or cellular remodelling. Similar to other MLCK-like kinases like DRAK2 and DAPK1, TK is linked to protein turnover regulation via the autophagy/lysosomal system, suggesting the MLCK-like kinases have common functions beyond contraction regulation

TITINdb:A computational tool to assess titin's role as a disease gene

Large numbers of rare and unique titin missense variants have been discovered in both healthy and disease cohorts, thus the correct classification of variants as pathogenic or non-pathogenic has become imperative. Due to titin's large size (363 coding exons), current web applications are unable to map titin variants to domain structures. Here, we present a web application, TITINdb, which integrates titin structure, variant, sequence and isoform information, along with pre-computed predictions of the impact of non-synonymous single nucleotide variants, to facilitate the correct classification of titin variants

Current and future therapeutic approaches to the congenital myopathies

The congenital myopathies − including Central Core Disease (CCD), Multi-minicore Disease (MmD), Centronuclear Myopathy (CNM), Nemaline Myopathy (NM) and Congenital Fibre Type Disproportion (CFTD) − are a genetically heterogeneous group of early-onset neuromuscular conditions characterized by distinct histopathological features, and associated with a substantial individual and societal disease burden. Appropriate supportive management has substantially improved patient morbidity and mortality but there is currently no cure. Recent years have seen an exponential increase in the genetic and molecular understanding of these conditions, leading to the identification of underlying defects in proteins involved in calcium homeostasis and excitation-contraction coupling, thick/thin filament assembly and function, redox regulation, membrane trafficking and/or autophagic pathways. Based on these findings, specific therapies are currently being developed, or are already approaching the clinical trial stage. Despite undeniable progress, therapy development faces considerable challenges, considering the rarity and diversity of specific conditions, and the size and complexity of some of the genes and proteins involved. The present review will summarize the key genetic, histopathological and clinical features of specific congenital myopathies, and outline therapies already available or currently being developed in the context of known pathogenic mechanisms. The relevance of newly discovered molecular mechanisms and novel gene editing strategies for future therapy development will be discussed

Myosin binding protein-C activates thin filaments and inhibits thick filaments in heart muscle cells

Myosin binding protein-C (MyBP-C) is a key regulatory protein in heart muscle, and mutations in the MYBPC3 gene are frequently associated with cardiomyopathy. However, the mechanism of action of MyBP-C remains poorly understood, and both activating and inhibitory effects of MyBP-C on contractility have been reported. To clarify the function of the regulatory N-terminal domains of MyBP-C, we determined their effects on the structure of thick (myosin-containing) and thin (actin-containing) filaments in intact sarcomeres of heart muscle. We used fluorescent probes on troponin C in the thin filaments and on myosin regulatory light chain in the thick filaments to monitor structural changes associated with activation of demembranated trabeculae from rat ventricle by the C1mC2 region of rat MyBP-C. C1mC2 induced larger structural changes in thin filaments than calcium activation, and these were still present when active force was blocked with blebbistatin, showing that C1mC2 directly activates the thin filaments. In contrast, structural changes in thick filaments induced by C1mC2 were smaller than those associated with calcium activation and were abolished or reversed by blebbistatin. Low concentrations of C1mC2 did not affect resting force but increased calcium sensitivity and reduced cooperativity of force and structural changes in both thin and thick filaments. These results show that the N-terminal region of MyBP-C stabilizes the ON state of thin filaments and the OFF state of thick filaments and lead to a novel hypothesis for the physiological role of MyBP-C in the regulation of cardiac contractility.</p

Epigenetic changes as a common trigger of muscle weakness in congenital myopathies

Congenital myopathies are genetically and clinically heterogeneous conditions causing severe muscle weakness, and mutations in the ryanodine receptor gene (RYR1) represent the most frequent cause of these conditions. A common feature of diseases caused by recessive RYR1 mutations is a decrease of ryanodine receptor 1 protein content in muscle. The aim of the present investigation was to gain mechanistic insight into the causes of this reduced ryanodine receptor 1. We found that muscle biopsies of patients with recessive RYR1 mutations exhibit decreased expression of muscle-specific microRNAs, increased DNA methylation and increased expression of class II histone deacetylases. Transgenic mouse muscle fibres over-expressing HDAC-4/HDAC-5 exhibited decreased expression of RYR1 and of muscle-specific miRNAs, whereas acute knock-down of RYR1 in mouse muscle fibres by siRNA caused up-regulation of HDAC-4/HDAC-5. Intriguingly, increased class II HDAC expression and decreased ryanodine receptor protein and miRNAs expression were also observed in muscles of patients with nemaline myopathy, another congenital neuromuscular disorder. Our results indicate that a common pathophysiological pathway caused by epigenetic changes is activated in some forms of congenital neuromuscular disorder

Developmental regulation of MURF E3 ubiquitin ligases in skeletal muscle

The striated muscle-specific tripartite motif (TRIM) proteins TRIM63/MURF1, TRIM55/MURF2 and TRIM54/MURF3 can function as E3 ubiquitin ligases in ubiquitin-mediated muscle protein turnover. Despite the well-characterised role of MURF1 in skeletal muscle atrophy, the dynamics of MURF isogene expression in the development and early postnatal adaptation of skeletal muscle is unknown. Here, we show that MURF2 is the isogene most highly expressed in embryonic skeletal muscle at E15.5, with the 50 kDa A isoform predominantly expressed. MURF1 and MURF3 are upregulated only postnatally. Knockdown of MURF2 p50A by isoform-specific siRNA results in delayed myogenic differentiation and myotube formation in vitro, with perturbation of the stable, glutamylated microtubule population. This underscores that MURF2 plays an important role in the earliest stages of skeletal muscle differentiation and myofibrillogenesis. During further development, there is a shift towards the 60 kDa A isoform, which dominates postnatally. Analysis of the fibre-type expression shows that MURF2 A isoforms are predominantly slow-fibre associated, whilst MURF1 is largely excluded from these fibres, and MURF3 is ubiquitously distributed in both type I and II fibres.</p

Sub-diffraction error mapping for localisation microscopy images

Assessing the quality of localisation microscopy images is highly challenging due to the difficulty in reliably detecting errors in experimental data. The most common failure modes are the biases and errors produced by the localisation algorithm when there is emitter overlap. Also known as the high density or crowded field condition, significant emitter overlap is normally unavoidable in live cell imaging. Here we use Haar wavelet kernel analysis (HAWK), a localisation microscopy data analysis method which is known to produce results without bias, to generate a reference image. This enables mapping and quantification of reconstruction bias and artefacts common in all but low emitter density data. By avoiding comparisons involving intensity information, we can map structural artefacts in a way that is not adversely influenced by nonlinearity in the localisation algorithm. The HAWK Method for the Assessment of Nanoscopy (HAWKMAN) is a general approach which allows for the reliability of localisation information to be assessed

Vici syndrome: a review

Vici syndrome [OMIM242840] is a severe, recessively inherited congenital disorder characterized by the principal features of callosal agenesis, cataracts, oculocutaneous hypopigmentation, cardiomyopathy, and a combined immunodeficiency. Profound developmental delay, progressive failure to thrive and acquired microcephaly are almost universal, suggesting an evolving (neuro) degenerative component. In most patients there is additional variable multisystem involvement that may affect virtually any organ system, including lungs, thyroid, liver and kidneys. A skeletal myopathy is consistently associated, and characterized by marked fibre type disproportion, increase in internal nuclei, numerous vacuoles, abnormal mitochondria and glycogen storage. Life expectancy is markedly reduced.Vici syndrome is due to recessive mutations in EPG5 on chromosome 18q12.3, encoding ectopic P granules protein 5 (EPG5), a key autophagy regulator in higher organisms. Autophagy is a fundamental cellular degradative pathway conserved throughout evolution with important roles in the removal of defective proteins and organelles, defence against infections and adaptation to changing metabolic demands. Almost 40 EPG mutations have been identified to date, most of them truncating and private to individual families.The differential diagnosis of Vici syndrome includes a number of syndromes with overlapping clinical features, neurological and metabolic disorders with shared CNS abnormalities (in particular callosal agenesis), and primary neuromuscular disorders with a similar muscle biopsy appearance. Vici syndrome is also the most typical example of a novel group of inherited neurometabolic conditions, congenital disorders of autophagy.Management is currently largely supportive and symptomatic but better understanding of the underlying autophagy defect will hopefully inform the development of targeted therapies in future.</p

Structure of the native myosin filament in the relaxed cardiac sarcomere

The sarcomere is the basic unit of striated muscles and consists of interdigitating thick and thin filaments. The two types of filaments slide on each other resulting in the shortening of sarcomere itself thereby generating work. The thin filament comprises filamentous actin (F-actin), troponin (Tn), and tropomyosin (Tm). The thick filament is the force bearing part of the sarcomere and it comprises myosin, titin, and myosin-binding protein C (MyBP-C). The vast majority of the mutations responsible for familial hypertrophic cardiomyopathy and other heart and muscle diseases are borne by components of the thick filaments. However, despite its central importance, it remains unclear how thick filaments are structurally organized and how its components interact with each other and with thin filaments to enable highly regulated muscle contraction in the cardiac tissue. In this thesis, I aimed to elucidate the molecular organization of the thick and thin filaments from left ventricular myofibrils in their relaxed state. I resorted to cryo-focused ion beam milling (cryo-FIB- milling) and cryo-electron tomography (cryo-ET) to investigate the molecular architecture of the native mammalian cardiac sarcomeres. The reconstruction of the thick filament provides insight into the three-dimensional arrangement of myosin heads and tails, MyBP-C, and titin. This clarification of structural details sheds light on their interplay during muscle contraction. The positioning of myosin heads exhibits variability based on their location along the filament, indicating diverse capabilities in terms of susceptibility to strain and activation. Meanwhile, the arrangement and interactions of myosin tails follow a distinct pattern, potentially governed by the organization of three alpha and three beta titin chains resembling a spring. This hints at specialized functions of thick filament segments in length-dependent activation (LDA) and contraction. Interestingly, the three titin alpha chains extend throughout the entire length of the thick filament, in contrast to titin beta. The structural analysis further reveals that the C-terminal region of MyBP-C not only binds to myosin tails but also unexpectedly interacts directly with myosin heads. This suggests a hitherto unreported direct involvement in maintaining the myosin OFF state. Furthermore, we visualize how MyBP-C forms links between thin and thick filaments. This study provides a molecular blueprint of the cardiac sarcomere and paves the way to forthcoming research aiming to explore muscle disorders that involve sarcomeric structural components.Das Sarkomer ist die Grundeinheit der quergestreiften Muskulatur und besteht aus ineinandergreifenden dicken und dünnen Filamenten. Die Filamente verschieben sich ineinander, was zur Verkürzung des Sarkomers führt und dadurch Kraft erzeugt. Das dünne Filament besteht aus filamentösem Aktin (F-Actin), Troponin (Tn) und Tropomyosin (Tm). Das dicke Filament ist der krafttragende Teil des Sarkomers und besteht aus Myosin, Titin und dem Myosin-bindenden Protein C (MyBP-C). Die überwiegende Mehrheit der Mutationen, die für die familiäre hypertrophe Kardiomyopathie und andere Herz- und Muskelerkrankungen verantwortlich sind, finden sich in Komponenten des dicken Filaments. Trotz ihrer zentralen Bedeutung ist jedoch nach wie vor unklar, wie die dicken Filamente strukturell organisiert sind und wie ihre Komponenten miteinander und mit den dünnen Filamenten interagieren, um eine regulierte Muskelkontraktion im Herzgewebe zu ermöglichen. In dieser Arbeit habe ich das Ziel verfolgt, die molekulare Organisation der dicken und dünnen Filamente von ventrikulären Myofibrillen im entspannten Zustand aufzuklären. Dazu nutzte ich einen fokussierten Ionenstrahl bei Kryo-Bedingungen, um die Proben vorzubereiten (cryo-FIB-milling) und Kryo-Elektronentomographie (cryo-ET), um die molekulare Architektur der nativen Herzsarkomere von Säugetieren zu betimmen. Die Rekonstruktion des dicken Filaments zeigt die dreidimensionale Organisation der Myosinköpfe und -schwänze, des MyBP-C und Titins und klärt die strukturelle Grundlage für ihre Interaktion während der Muskelkontraktion auf. Die Anordnung der Myosinköpfe ist je nach ihrer Position entlang des Filaments variabel, was darauf hindeutet, dass sie unterschiedliche Fähigkeiten in Bezug auf Belastungsanfälligkeit und Aktivierung haben. Auch die Myosinschwänze weisen eine unterschiedliche Anordnung und ein unterschiedliches Muster von Interaktionen auf. Diese werden wahrscheinlich von drei alpha- und drei beta-Titinketten orchestriert, die wie eine Feder angeordnet sind. Dies deutet darauf hin, dass unterschiedliche Segmente des dicken Filaments bei der längenabhängigen Aktivierung und Kontraktion eine besondere Rolle spielen. Überraschenderweise verlaufen die drei alpha-Titinketten über die gesamte Länge des dicken Filaments, die beta-Titinkette jedoch nicht. Die Struktur zeigt auch, dass die C-terminale Region von MyBP-C Myosin-Schwänze bindet und unerwarteterweise auch direkt mit den Myosin-Köpfen interagiert, was auf eine bisher unbeschriebene direkte Rolle bei der Aufrechterhaltung des Myosin- OFF-Zustands hindeutet. Darüber hinaus konnte ich zeigen, wie MyBP-C Verbindungen zwischen dünnen und dicken Filamenten bildet. Meine Arbeit liefert einen molekularen Bauplan des Herzsarkomers und ebnet den Weg für künftige Forschungsarbeiten zur Erforschung von Muskelerkrankungen, an denen strukturelle Komponenten des Sarkomers beteiligt si