Muscle Thin Filaments Research Articles

Characterization of NEB pathogenic variants in patients reveals novel nemaline myopathy disease mechanisms and omecamtiv mecarbil force effects.

Nebulin, a critical protein of the skeletal muscle thin filament, plays important roles in physiological processes such as regulating thin filament length (TFL), cross-bridge cycling, and myofibril alignment. Pathogenic variants in the nebulin gene (NEB) cause NEB-based nemaline myopathy (NEM2), a genetically heterogeneous disorder characterized by hypotonia and muscle weakness, currently lacking curative therapies. In this study, we examined a cohort of ten NEM2 patients, each with unique pathogenic variants, aiming to understand their impact on mRNA, protein, and functional levels. Results show that pathogenic truncation variants affect NEB mRNA stability and lead to nonsense-mediated decay of the mutated transcript. Moreover, a high incidence of cryptic splice site activation was found in patients with pathogenic splicing variants that are expected to disrupt the actin-binding sites of nebulin. Determination of protein levels revealed patients with either relatively normal or markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in TFL, or reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a pathogenic duplication variant in nebulin that resulted in a four-copy gain in the triplicate region of NEB and a much larger nebulin protein and longer TFL. Additionally, we investigated the effect of Omecamtiv mecarbil (OM), a small-molecule activator of cardiac myosin, on force production of type 1 muscle fibers of NEM2 patients. OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87 to 318%, with the largest effects in patients with the lowest level of nebulin. In summary, this study indicates that post-transcriptional or post-translational mechanisms regulate nebulin expression. Moreover, we propose that the pathomechanism of NEM2 involves not only shortened but also elongated thin filaments, along with the disruption of actin-binding sites resulting from pathogenic splicing variants. Significantly, our findings highlight the potential of OM treatment to improve skeletal muscle function in NEM2 patients, especially those with large reductions in nebulin levels.

Comprehensive phenotypic characterization of an allelic series of zebrafish models of NEB-related nemaline myopathy.

Nemaline myopathy (NM) is a rare congenital neuromuscular disorder characterized by muscle weakness and hypotonia, slow gross motor development, and decreased respiratory function. Mutations in at least twelve genes, all of each encode proteins that are either components of the muscle thin filament or regulate its length and stability, have been associated with NM. Mutations in Nebulin (NEB), a giant filamentous protein localized in the sarcomere, account for more than 50% of NM cases. At present, there remains a lack of understanding of whether NEB genotype influences nebulin function and NM-patient phenotypes. In addition, there is a lack of therapeutically tractable models that can enable drug discovery and address the current unmet treatment needs of patients. To begin to address these gaps, here we have characterized five new zebrafish models of NEB-related NM. These mutants recapitulate most aspects of NEB-based NM, showing drastically reduced survival, defective muscle structure, reduced contraction force, shorter thin filaments, presence of electron-dense structures in myofibers, and thickening of the Z-disks. This study represents the first extensive investigation of an allelic series of nebulin mutants, and thus provides an initial examination in pre-clinical models of potential genotype-phenotype correlations in human NEB patients. It also represents the first utilization of a set of comprehensive outcome measures in zebrafish, including correlation between molecular analyses, structural and biophysical investigations, and phenotypic outcomes. Therefore, it provides a rich source of data for future studies exploring the NM pathomechanisms, and an ideal springboard for therapy identification and development for NEB-related NM.

Abstract EC.09: Single Nuclei RNA Sequencing Analysis Of Mybpc-3- Associated HCM To Identify Common Pathological Mechanisms In Human, Feline, And Murine Hearts

Hypertrophic cardiomyopathy (HCM) is a common inherited cardiovascular disease characterized by unexplained hypertrophy, often associated with left ventricular outflow tract (LVOT) obstruction, ventricular tachyarrhythmias, diastolic heart failure, and sudden cardiac death. Mutations in mybpc3 are the most common genetic mutations associated with human and feline HCM. To understand how mutations in mybc3 causing sarcomere dysfunction result in the development of non-myocyte phenotypes such as fibrosis, mitral valve anomalies, and microvascular occlusion across species, we generated single nuclei RNA-sequencing datasets from feline, human and murine heart tissue carrying mybpc3 variants. We analyzed these datasets for differential gene expression by genotype for each cell type and assessed changes in cell-cell communication by ligand-receptor analysis. Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs) in feline cardiomyocytes revealed alteration in regulation of calcium ion transmembrane transporter activity, regulation of cation channel activity, and extracellular matrix structure and organization, consistent with known phenotypic aspects of HCM such as calcium dyshomeostasis and fibrosis. DEGs in cardiomyocytes of human tissues revealed novel obstruction of cGMP-mediated signaling, protein O-linked glycosylation and negative regulation of cAMP-mediated signaling. DEGs in murine cardiomyocytes affected cardiac muscle thin filament assembly, CDP-diacylglycerol biosynthesis and cell adhesion molecule production. Analysis of intercellular communication in feline and murine hearts revealed an overall loss of ligands broadcast from all cell types, with notable losses from fibroblast cells and cardiomyocytes. These findings are consistent with recent studies implicating altered intercellular communication between cardiomyocytes and non-myocyte cell types as a potential disease mechanism in human HCM. We will further identify common and species-specific disease-promoting pathways in HCM tissue that will allow identification of conserved disease mechanisms that may be targeted in both human and feline disease and will facilitate an understanding of the relevance of mouse models.

A new tool for producing and investigating recombinant sarcomeric actin.

Actin forms many cytostructural elements, including the backbone of striated muscle thin filaments. Our mechanistic understanding of most actin-dependent processes remains limited, partly due to difficulty with recombinant actin expression. While fully-functional actin cannot be produced in bacteria, it can be generated using baculovirus expression systems. Here, we created an additional tool for recombinant actin production, for hypothesis testing and for resolving the molecular properties and cellular effects of unique variants. Specifically, we engineered Drosophila that can express untagged actins exclusively in indirect flight muscles (IFMs). Act88F encodes sarcomeric IFM actin. Attempts to rescue IFM function (i.e. flight) in Act88F-nulls, by inserting transgenic Act88F (Act88FTG-WT) ectopically throughout the genome have failed, potentially due to ineffective cis/trans regulation of Act88FTG-WT expression. To circumvent these problems, we used CRISPR-editing and introduced an attP landing site directly into Act88F, to disrupt the endogenous gene and serve as a target for site-specific transgene insertion. attP-mediated disruption of Act88F yielded flightlessness and actin-free IFMs. Transformants expressing GFP or Act88FTG-WT, from the endogenous Act88F integration locus, demonstrated robust IFM-specific protein production and restored flight, respectively. Having established the expression system, we designed Drosophila mutants to test the roles of specific actin residues in tropomyosin/myosin-binding and nemaline myopathy pathogenesis. Both Act88FTG-R147Q and Act88F TG-F352Striggered IFM hypercontraction and impaired flight. Finally, we determined whether mammalian actin could be generated and observed high-level expression of human α-cardiac actin (ACTC1), which behaved indistinguishably from bovine ACTC1 in unregulated and regulated motility (IVM) assays. We are currently comparing IVM properties of IFM-purified vs. baculovirus/Sf12-purified ACTC1. Overall, our novel fly line permits transgenic actin expression in mature muscle fibers that are themselves amenable to structural and functional analyses, and serves as a cost-effective, perpetual source of ample protein for biophysical experimentation.

Rumen Epithelial Development- and Metabolism-Related Genes Regulate Their Micromorphology and VFAs Mediating Plateau Adaptability at Different Ages in Tibetan Sheep.

The rumen is an important hallmark organ of ruminants and plays an important role in the metabolism and immune barrier of Tibetan sheep on the Plateau. However, there are few studies on rumen development and metabolism regulation in Tibetan sheep at different ages. Here, we comprehensively analyzed the immune function, fermentation function, rumen epithelial micromorphology and transcriptome profile of Tibetan sheep at different ages. The results showed that the concentration of IgG decreased and the concentration of IgM increased with age (p < 0.05), and the highest concentration of IgA was observed at 1.5 and 3.5 years of age. In terms of rumen fermentation characteristics, VFAs of 4-month-old lambs were the highest, followed by VFAs and NH3-N of Tibetan sheep at 3.5 years of age. Hematoxylin-eosin staining and transmission electron microscopy section examination of rumen epithelial tissue showed that the rumen papilla width increased with age (p < 0.001), the thickness of the stratum corneum decreased, the cells in the stratum corneum showed accelerated migration and the thickness of the rumen muscle layer increased (p < 0.001). Desmosomal junctions between the layers of rumen epithelium increased at 1.5 and 3.5 years old, forming a compact barrier structure, and the basal layer had more mitochondria involved in the regulation of energy metabolism. RNA-seq analysis revealed that a total of 1006 differentially expressed genes (DEGs) were identified at four ages. The DEGs of Tibetan sheep aged 4 months and 6 years were mainly enriched in the oxidation−reduction process and ISG15-protein conjugation pathway. The 1.5 and 3.5-year-olds were mainly enriched in skeletal muscle thin filament assembly, mesenchyme migration and the tight junction pathway. WGCNA showed that DEGs related to rumen microbiota metabolite VFAs and epithelial morphology were enriched in “Metabolism of xenobiotics by cytochrome P450, PPAR signaling pathway, Butanoate metabolism pathways” and participated in the regulation of rumen epithelial immune and fermentation metabolism functions of Tibetan sheep at different ages. This study systematically revealed the regulatory mechanism of rumen epithelial development and metabolism in the plateau adaptation of Tibetan sheep, providing a new approach for the study of plateau adaptation.

Cyclophosphamide treatment modifies the thermal stability of profilin bound monomeric and leiomodin2 bound filamentous actin

The monomeric (G-actin) and polymer (F-actin) forms of actin play important role in muscle development and contraction, cellular motility, division, and transport processes. Leiomodins 1–3 (Lmod1–3) are crucial for the development of muscle sarcomeres. Unlike tropomodulins that localize only at the pointed ends, the striated muscle specific Lmod2 shows diffuse distribution along the entire length of the thin filaments. The G-actin-binding profilin (Pro) facilitates the nucleotide exchange on monomeric actin and inhibits the polymerization at the barbed end, therefore contributes to the maintenance of the intracellular pool of polymerization competent ATP-G-actin. Cyclophosphamide (CP) is a cytostatic drug that can have potential side effects on muscle thin filaments at the level of actin in myofilaments. Here, we aimed at investigating the influence of CP on actin and its complexes with actin-binding proteins by using differential scanning calorimetry (DSC). We found that upon CP treatment, the denaturation of the Pro-G-actin and Lmod2-F-actin complexes was characterized by an increased enthalpy change. However, after the CP treatment, the melting temperature of F-actin was the same as in the presence of Lmod2, seems like Lmod2 does not have any effect on the structure of the CP alkylated F-actin. In case of Pro bound G-actin the melting temperature did not respond to the CP addition. The intracellular function of Lmod2 in muscle cells can be modified within CP drug treatment.

The Central Role of the F-Actin Surface in Myosin Force Generation.

Simple SummaryAlthough actin is a highly conserved protein, it is involved in many diverse cellular processes. Actin owes its diversity of function to its ability to bind to a host of actin-binding proteins (ABPs) that localize across its surface. Among the most studied ABPs is the molecular motor, myosin. Myosin generates force on actin filaments by pairing ATP hydrolysis, product release, and actin-binding to the conformational changes that lead to movement. Central to this process is the progression of myosin binding to the actin surface as it moves through its ATPase cycle. During binding, actin acts as a myosin ATPase activator, catalyzing essential hydrolysis release steps. Here, we use the current model of actin-myosin binding as a roadmap to describe the portions of the actin-myosin interface that are sequentially formed throughout the motor cycle. At each step, we compare the interactions of a diverse set of high-resolution actin-myosin cryo-electron microscopy structures to define what portions of the interface are conserved and which are isoform-specific.Actin is one of the most abundant and versatile proteins in eukaryotic cells. As discussed in many contributions to this Special Issue, its transition from a monomeric G-actin to a filamentous F-actin form plays a critical role in a variety of cellular processes, including control of cell shape and cell motility. Once polymerized from G-actin, F-actin forms the central core of muscle-thin filaments and acts as molecular tracks for myosin-based motor activity. The ATP-dependent cross-bridge cycle of myosin attachment and detachment drives the sliding of myosin thick filaments past thin filaments in muscle and the translocation of cargo in somatic cells. The variation in actin function is dependent on the variation in muscle and non-muscle myosin isoform behavior as well as interactions with a plethora of additional actin-binding proteins. Extensive work has been devoted to defining the kinetics of actin-based force generation powered by the ATPase activity of myosin. In addition, over the past decade, cryo-electron microscopy has revealed the atomic-evel details of the binding of myosin isoforms on the F-actin surface. Most accounts of the structural interactions between myosin and actin are described from the perspective of the myosin molecule. Here, we discuss myosin-binding to actin as viewed from the actin surface. We then describe conserved structural features of actin required for the binding of all or most myosin isoforms while also noting specific interactions unique to myosin isoforms.

A Potent Fluorescent Reversible-Covalent Inhibitor of Cardiac Muscle Contraction.

Compounds that directly modulate the response of the cardiac sarcomere have potential in the treatment of cardiac disease. While a number of sarcomere activators have been discovered and extensively studied, very few inhibitors have been identified. We report a potent cardiac sarcomere inhibitor, DN-F01, targeting the cardiac muscle thin filament protein troponin complex. Functional studies show that DN-F01 has a strong inhibitory calcium-dependent effect on cardiac myofibrillar ATPase activity with an IC50 value of 11 ± 4 nmol/L. DN-F01 is shown to bind to a cardiac troponin C-troponin I chimera (cChimera) with a K D of ∼50 nM using fluorescence spectroscopy, indicating that troponin is the likely target for DN-F01. NMR titrations of DN-F01 to C35S and A-Cys cChimera show covalent and noncovalent binding of DN-F01 bound to the calcium-saturated cChimera.

Post-translational protein deimination signatures in sea lamprey (Petromyzon marinus) plasma and plasma-extracellular vesicles

Lampreys are a jawless vertebrate species belonging to an ancient vertebrate lineage that diverged from a common ancestor with humans ~500 million years ago. The sea lamprey (Petromyzon marinus) has a filter feeding ammocoete larval stage that metamorphoses into a parasitic adult, feeding both on teleost and elasmobranch fish. Lampreys are a valuable comparative model species for vertebrate immunity and physiology due to their unique phylogenetic position, unusual adaptive immune system, and physiological adaptions such as tolerance to salinity changes and urea.Peptidylarginine deiminases (PADs) are a phylogenetically conserved enzyme family which catalyses post-translational deimination/citrullination in target proteins, enabling proteins to gain new functions (moonlighting). The identification of deiminated protein targets in species across phylogeny may provide novel insights into post-translational regulation of physiological and pathophysiological processes. Extracellular vesicles (EVs) are membrane vesicles released from cells that carry cargos of small molecules and proteins for cellular communication, involved in both normal and pathological processes. The current study identified deimination signatures in proteins of both total plasma and plasma-EVs in sea lamprey and furthermore reports the first characterisation of plasma-EVs in lamprey. EVs were poly-dispersed in the size range of 40–500 nm, similar to what is observed in other taxa, positive for CD63 and Flotillin-1. Plasma-EV morphology was confirmed by transmission electron microscopy. Assessment of deimination/citrullination signatures in lamprey plasma and plasma-EVs, revealed 72 deimination target proteins involved in immunity, metabolism and gene regulation in whole plasma, and 37 target proteins in EVs, whereof 24 were shared targets. Furthermore, the presence of deiminated histone H3, indicative of gene-regulatory mechanisms and also a marker of neutrophil extracellular trap formation (NETosis), was confirmed in lamprey plasma. Functional protein network analysis revealed some differences in KEGG and GO pathways of deiminated proteins in whole plasma compared with plasma-EVs. For example, while common STRING network clusters in plasma and plasma-EVs included Peptide chain elongation, Viral mRNA translation, Glycolysis and gluconeogenesis, STRING network clusters specific for EVs only included: Cellular response to heat stress, Muscle protein and striated muscle thin filament, Nucleosome, Protein processing in endoplasmic reticulum, Nucleosome and histone deacetylase complex. STRING network clusters specific for plasma were: Adipokinetic hormone receptor activity, Fibrinogen alpha/beta chain family, peptidase S1A, Glutathione synthesis and recycling-arginine, Fructose 1,6-bisphosphate metabolic process, Carbon metabolism and lactate dehydrogenase activity, Post-translational protein phosphorylation, Regulation of insulin-like growth factor transport and clotting cascade. Overall, for the EV citrullinome, five STRING network clusters, 10 KEGG pathways, 15 molecular GO pathways and 29 Reactome pathways were identified, compared with nine STRING network clusters, six KEGG pathways, two Molecular GO pathways and one Reactome pathway specific for whole plasma; while further pathways were shared. The reported findings indicate that major pathways relevant for immunity and metabolism are targets of deimination in lamprey plasma and plasma-EVs, with some differences, and may help elucidating roles for the conserved PAD enzyme family in regulation of immune and metabolic function throughout phylogeny.

Molecular dynamics provides new insights into the mechanism of calcium signal transduction and interdomain interactions in cardiac troponin.

Understanding the regulation of cardiac muscle contraction at a molecular level is crucial for the development of therapeutics for heart conditions. Despite the availability of atomic structures of the protein components of cardiac muscle thin filaments, detailed insights into their dynamics and response to calcium are yet to be fully depicted. In this study, we used molecular dynamics simulations of the core domains of the cardiac muscle protein troponin to characterize the equilibrium dynamics of its calcium-bound and calcium-free forms, with a focus on elements of cardiac muscle contraction activation and deactivation, that is, calcium binding to the cardiac troponin Ca2+ -binding subunit (TnC) and the release of the switch region of the troponin inhibitory subunit (TnI) from TnC. The process of calcium binding to the TnC binding site is described as a three-step process commencing with calcium capture by the binding site residues, followed by cooperative residue interplay bringing the calcium ion to the binding site, and finally, calcium-water exchange. Furthermore, we uncovered a set of TnC-TnI interdomain interactions that are critical for TnC N-lobe hydrophobic pocket dynamics. Absence of these interactions allows the closure of the TnC N-lobe hydrophobic pocket while the TnI switch region remains expelled, whereas if the interactions are maintained, the hydrophobic pocket remains open. Modification of these interactions may fine-tune the ability of the TnC N-lobe hydrophobic pocket to close or remain open, modulate cardiac contractility and present potential therapy-relevant targets.

C-terminal troponin-I residues trap tropomyosin in the muscle thin filament blocked-state

Tropomyosin and troponin regulate muscle contraction by participating in a macromolecular scale steric-mechanism to control myosin-crossbridge – actin interactions and consequently contraction. At low-Ca2+, the C-terminal 30% of troponin subunit-I (TnI) is proposed to trap tropomyosin in a position on thin filaments that sterically interferes with myosin-binding, thus causing muscle relaxation. In contrast, at high-Ca2+, inhibition is released after the C-terminal domains dissociate from F-actin-tropomyosin as its component switch-peptide domain binds to the N-lobe of troponin-C (TnC). Recent, paradigm-shifting, cryo-EM reconstructions by the Namba group have revealed density attributed to TnI along cardiac muscle thin filaments at both low- and high-Ca2+ concentration. Modeling the reconstructions showed expected high-Ca2+ hydrophobic interactions of the TnI switch-peptide and TnC. However, under low-Ca2+ conditions, sparse interactions of TnI and tropomyosin, and in particular juxtaposition of non-polar switch-peptide residues and charged tropomyosin amino acids in the published model seem difficult to reconcile with an expected steric-blocking conformation. This anomaly is likely due to inaccurate fitting of tropomyosin into the cryo-EM volume. In the current study, the low-Ca2+ cryo-EM volume was fitted with a more accurate tropomyosin model and representation of cardiac TnI. Our results show that at low-Ca2+ a cluster of hydrophobic residues at the TnI switch-peptide and adjacent H4 helix (Ala149, Ala151, Met 154, Leu159, Gly160, Ala161, Ala163, Leu167, Leu169, Ala171, Leu173) draw-in tropomyosin surface residues (Ile143, Ile146, Ala151, Ile154), presumably attracting the entire tropomyosin cable to its myosin-blocking position on actin. The modeling confirms that neighboring TnI “inhibitory domain” residues (Arg145, Arg148) bind to thin filaments at actin residue Asp25, as previously suggested. ClusPro docking of TnI residues 137–184 to actin-tropomyosin, including the TnI inhibitory-domain, switch-peptide and Helix H4, verified the modeled configuration. Our residue-to-residue contact-mapping of the TnI-tropomyosin association lends itself to experimental validation and functional localization of disease-bearing mutations.

Genetically Engineered Addition of a Ca2+ Buffering Capacity in Mouse Cardiac Muscle Thin Filament using a Glu-Rich Segment of Insect Troponin T Alters Cardiac Performance

In addition to a core function in Ca2+ regulation of muscle contraction, insect troponin T (TnT) has a unique glutamic acid-rich long C-terminal extension. With sequence similarity to this diverged structure, the N-terminal segment of avian flight muscle TnT binds Ca2+ with a physiologically relevant affinity (Zhang et al., Biochemistry 43:2645-55, 2004). Deletion of the C-terminal extension of Drosophila TnT significantly decreased muscle and heart functions (Cao et al., JBC 295:3794-3807, 2020). We propose a hypothesis that this structure serves as a myofilament Ca2+ reservoir to reduce the amount of Ca2+ cycled by Ca2+ pumps during muscle contraction and relaxation. A CRISP/Cas9 knock-in (KI) mouse model was constructed to replace the N-terminal variable region of cardiac TnT with 51 Glu residues derived from the C-terminal extension of bee TnT. Cardiac function is studied using ex vivo working hearts and tested for responses to beta-adrenergic stimulation or L-Ca2+ channel agonist Bay-K8644. At baseline, cTnT-51E hearts showed lower systolic velocity than that of WT control. Upon isoproterenol or Bay-K8644 treatment, cTnT-51E and wild type hearts both had positive responses in performance. However, cTnT-51E hearts exhibited significantly more increases in ventricular peak systolic pressure and diastolic velocity but still lower stroke volume as compared to that of WT controls. This phenotype is likely due to an altered response to the increase in intracellular calcium. Further analysis of Ca2+ responses of skinned muscle sections, cardiac energetic efficiency and intracellular Ca2+ transient are underway to establish the novel Ca2+ buffering capacity of TnT and its function in contractile kinetics of striated muscle.

Binding of calcium and magnesium to human cardiac troponin C

Cardiac muscle thin filaments are composed of actin, tropomyosin, and troponin that change conformation in response to Ca2+ binding, triggering muscle contraction. Human cardiac troponin C (cTnC) is the Ca2+-sensing component of the thin filament. It contains structural sites (III/IV) that bind both Ca2+ and Mg2+ and a regulatory site (II) that has been thought to bind only Ca2+. Binding of Ca2+ at this site initiates a series of conformational changes that culminate in force production. However, the mechanisms that underpin the regulation of binding at site II remain unclear. Here, we have quantified the interaction between site II and Ca2+/Mg2+ through isothermal titration calorimetry and thermodynamic integration simulations. Direct and competitive binding titrations with WT N-terminal cTnC and full-length cTnC indicate that physiologically relevant concentrations of both Ca2+/Mg2+ interacted with the same locus. Moreover, the D67A/D73A N-terminal cTnC construct in which two coordinating residues within site II were removed was found to have significantly reduced affinity for both cations. In addition, 1 mM Mg2+ caused a 1.4-fold lower affinity for Ca2+. These experiments strongly suggest that cytosolic-free Mg2+ occupies a significant population of the available site II. Interaction of Mg2+ with site II of cTnC likely has important functional consequences for the heart both at baseline as well as in diseased states that decrease or increase the availability of Mg2+, such as secondary hyperparathyroidism or ischemia, respectively.

Mechanical contribution to muscle thin filament activation

Vertebrate striated muscle thin filaments are thought to be thermodynamically activated in response to an increase in Ca2+ concentration. We tested this hypothesis by measuring time intervals for gliding runs and pauses of individual skeletal muscle thin filaments in cycling myosin motility assays. A classic thermodynamic mechanism predicts that if chemical potential is constant, transitions between runs and pauses of gliding thin filaments will occur at constant rate as given by a Poisson distribution. In this scenario, rate is given by the odds of a pause, and hence, run times between pauses fit an exponential distribution that slopes negatively for all observable run times. However, we determined that relative density of observed run times fits an exponential only at low Ca2+ levels that activate filament gliding. Further titration with Ca2+, or adding excess regulatory proteins tropomyosin and troponin, shifted the relative density of short run times to fit the positive slope of a gamma distribution, which derives from waiting times between Poisson events. Events that arise during a run and prevent the chance of ending a run for a random interval of time account for the observed run time distributions, suggesting that the events originate with cycling myosin. We propose that regulatory proteins of the thin filament require the mechanical force of cycling myosin to achieve the transition state for activation. During activation, combinations of cycling myosin that contribute insufficient activation energy delay deactivation.

Cardiac muscle thin filament structures reveal calcium regulatory mechanism

Contraction of striated muscles is driven by cyclic interactions of myosin head projecting from the thick filament with actin filament and is regulated by Ca2+ released from sarcoplasmic reticulum. Muscle thin filament consists of actin, tropomyosin and troponin, and Ca2+ binding to troponin triggers conformational changes of troponin and tropomyosin to allow actin-myosin interactions. However, the structural changes involved in this regulatory mechanism remain unknown. Here we report the structures of human cardiac muscle thin filament in the absence and presence of Ca2+ by electron cryomicroscopy. Molecular models in the two states built based on available crystal structures reveal the structures of a C-terminal region of troponin I and an N-terminal region of troponin T in complex with the head-to-tail junction of tropomyosin together with the troponin core on actin filament. Structural changes of the thin filament upon Ca2+ binding now reveal the mechanism of Ca2+ regulation of muscle contraction.

The Nebulin Family LIM and SH3 Proteins Regulate Postsynaptic Development and Function.

Neuronal dendrites have specialized actin-rich structures called dendritic spines that receive and integrate most excitatory synaptic inputs. The stabilization of dendrites and spines during neuronal maturation is essential for proper neural circuit formation. Changes in dendritic morphology and stability are largely mediated by regulation of the actin cytoskeleton; however, the underlying mechanisms remain to be fully elucidated. Here, we present evidence that the nebulin family members LASP1 and LASP2 play an important role in the postsynaptic development of rat hippocampal neurons from both sexes. We find that both LASP1 and LASP2 are enriched in dendritic spines, and their knockdown impairs spine development and synapse formation. Furthermore, LASP2 exerts a distinct role in dendritic arbor and dendritic spine stabilization. Importantly, the actin-binding N-terminal LIM domain and nebulin repeats of LASP2 are required for spine stability and dendritic arbor complexity. These findings identify LASP1 and LASP2 as novel regulators of neuronal circuitry.SIGNIFICANCE STATEMENT Proper regulation of the actin cytoskeleton is essential for the structural stability of dendrites and dendritic spines. Consequently, the malformation of dendritic structures accompanies numerous neurologic disorders, such as schizophrenia and autism. Nebulin family members are best known for their role in regulating the stabilization and function of actin thin filaments in muscle. The two smallest family members, LASP1 and LASP2, are more structurally diverse and are expressed in a broader array of tissues. While both LASP1 and LASP2 are highly expressed in the brain, little is currently known about their function in the nervous system. In this study, we demonstrate the first evidence that LASP1 and LASP2 are involved in the formation and long-term maintenance of dendrites and dendritic spines.

O.34Leiomodin-3 (LMOD3) deficiency affects contractile function and structure of fast muscle fibres

Leiomodin-3 (LMOD3), encoded by LMOD3, is a member of the tropomodulin protein family localising predominantly near striated muscle thin filament pointed ends. Recently, the critical role of LMOD3 for skeletal muscle function became apparent when mutations in this gene were found to cause a particularly severe form of nemaline myopathy characterised by neonatally lethal generalised muscle weakness. Currently, LMOD3 is thought to be essential for the structure of the thin filament in skeletal muscle, however, knowledge about LMOD3s function and the cause of weakness in LMOD3 patients is limited. We aim to shed light on LMOD3s role in skeletal muscle by investigating the ultrastructure and contractile properties of skeletal muscle from Lmod3 knock-out (KO) mice. Contractile mechanics of intact extensor digitorum longus (EDL) and soleus muscles as well as contractile performance of permeabilised single muscle fibres showed that fast type 2a and 2b fibres were affected by LMOD3 absence whereas slow type 1 fibres appeared to have preserved contractile function. Additionally, fibre type analysis of Lmod3 KO tibialis anterior muscle showed selective atrophy of 2b fibres and a shift towards a slower fibre typing profile. Our research suggests LMOD3 deficiency causes impaired skeletal muscle contraction in fast myofibers, whereas slow type 1 fibres are unaffected. Additionally, the selective atrophy of 2b fibres indicates that LMOD3 is critical for the biology of this fibre type. The present study highlights a novel role for LMOD proteins during muscle contraction, in particular in fast 2a and 2b myofibres. It remains to be established how these findings translate to LMOD3 patients, but it is possible that LMOD3 loss directly influence the contractile mechanics in LMOD3 patients resulting in muscle weakness.

Toxic effects of a methanolic coal dust extract on fish early life stage

Coal dust is a contaminant that impacts the terrestrial and aquatic environment with a complex mixture of chemicals, including PAHs and metals. This study aims to evaluate the toxic effect of a methanolic coal dust extract on a fish early life stage by analyzing phenotypic alterations, transcriptome changes, and mortality in zebrafish (ZF) embryos. ZF embryos were exposed to methanolic coal dust extract at 1–5000 mg·L-1 and monitored using bright field microscopy 24 and 48 hpf to determine malformations and mortality. In situ hybridization, RNA sequencing, and qRT-PCR were employed to identify transcriptome changes in malformed embryos. Three malformed phenotypes were generated in a dose-dependent manner. In situ hybridization analysis revealed brain, somite, dorsal cord, and heart tube development biomarker alterations. Gene expression profile analysis identified changes in genes related to structural constituent of muscle, calcium ion binding, actin binding, melanin metabolic process, muscle contraction, sarcomere organization, cardiac myofibril assembly, oxidation-reduction process, pore complex, supramolecular fiber, striated muscle thin filament, Z disc, and intermediate filament. This study shows, for the first time, the malformations generated by a mixture of pollutants from a methanolic coal dust extract on a fish early life stage, constituting a potential risk for normal embryonic development of other aquatic vertebrate organisms. Furthermore, we establish that phenotypes and changes in gene expression induced by the extract constitute a target for future studies about mechanical toxicity and their utility as sensitive tools in environmental risk assessments for biota and humans exposed to coal mining activities.

Nebulin stiffens the thin filament and augments cross-bridge interaction in skeletal muscle

Nebulin is a giant sarcomeric protein that spans along the actin filament in skeletal muscle, from the Z-disk to near the thin filament pointed end. Mutations in nebulin cause muscle weakness in nemaline myopathy patients, suggesting that nebulin plays important roles in force generation, yet little is known about nebulin's influence on thin filament structure and function. Here, we used small-angle X-ray diffraction and compared intact muscle deficient in nebulin (using a conditional nebulin-knockout, Neb cKO) with control (Ctrl) muscle. When muscles were activated, the spacing of the actin subunit repeat (27 Å) increased in both genotypes; when converted to thin filament stiffness, the obtained value was 30 pN/nm in Ctrl muscle and 10 pN/nm in Neb cKO muscle; that is, the thin filament was approximately threefold stiffer when nebulin was present. In contrast, the thick filament stiffness was not different between the genotypes. A significantly shorter left-handed (59 Å) thin filament helical pitch was found in passive and contracting Neb cKO muscles, as well as impaired tropomyosin and troponin movement. Additionally, a reduced myosin mass transfer toward the thin filament in contracting Neb cKO muscle was found, suggesting reduced cross-bridge interaction. We conclude that nebulin is critically important for physiological force levels, as it greatly stiffens the skeletal muscle thin filament and contributes to thin filament activation and cross-bridge recruitment.

Characterization of miRNA and their target gene during chicken embryo skeletal muscle development.

MicroRNAs (miRNAs) are non-coding RNAs that regulate mRNA expression by degradation or translational inhibition. We investigated the underlying molecular mechanisms of skeletal muscle development based on differentially expressed genes and miRNAs. We compared mRNA and miRNA from chicken skeletal muscle at embryonic day E11, E16 and one day post-hatch (P1). The interaction networks were constructed, according to target prediction results and integration analysis of up-regulated genes with down regulated miRNAs or down-regulated genes with up-regulated miRNAs with |log2fold change| ≥ 1.75, P < 0.005. The miRNA-mRNA integration analysis showed high number of mRNAs regulated by a few number of miRNAs. In the E11_VS_E16, comparison group we identified biological processes including muscle maintenance, myoblast proliferation and muscle thin filament formation. The E11_VS_P1 group comparison included negative regulation of axon extension, sarcomere organization, and cell redox homeostasis and kinase inhibitor activity. The E16_VS_P1 comparison group contained genes for the negative regulation of anti-apoptosis and axon extension as well as glomerular basement membrane development. Functional in vitro assays indicated that over expression of miR-222a and miR-126–5p in DF-1 cells significantly reduced the mRNA levels of the target genes CPEB3 and FGFR3, respectively. These integrated analyses provide several candidates for future studies concerning miRNAs-target function on regulation of embryonic muscle development and growth.