Sarcomeric Thin Filament Research Articles

An Update on Reported Variants in the Skeletal Muscle α‐Actin (ACTA1) Gene

The ACTA1 gene encodes skeletal muscle alpha‐actin, which forms the core of the sarcomeric thin filament in adult skeletal muscle. ACTA1 represents one of six highly conserved actin proteins that have all been associated with human disease. The first 15 pathogenic variants in ACTA1 were reported in 1999, which expanded to 177 in 2009. Here, we update on the now 607 total variants reported in LOVD, HGMD, and ClinVar, which includes 343 reported pathogenic/likely pathogenic (P/LP) variants. We also provide suggested ACTA1‐specific modifications to ACMG variant interpretation guidelines based on our analysis of known variants, gnomAD reports, and pathogenicity in other actin isoforms. Using these criteria, we report a total of 447 P/LP ACTA1 variants. From a clinical perspective, the number of reported ACTA1 disease phenotypes has grown from five to 20, albeit with some overlap. The vast majority (74%) of ACTA1 variants cause nemaline myopathy (NEM), but there are increasing numbers that cause cardiomyopathy and novel phenotypes such as distal myopathy. We highlight challenges associated with identifying genotype–phenotype correlations for ACTA1. Finally, we summarize key animal models and review the current state of preclinical treatments for ACTA1 disease. This update provides important resources and recommendations for the study and interpretation of ACTA1 variants.

Segregated localization of two calponin-related proteins within sarcomeric thin filaments in Caenorhabditis elegans striated muscle.

The calponin family proteins are expressed in both muscle and non-muscle cells and involved in the regulation of cytoskeletal dynamics and cell contractility. In the nematode Caenorhabditis elegans, UNC-87 and CLIK-1 are calponin-related proteins with 42% identical amino acid sequences containing seven calponin-like motifs. Genetic studies demonstrated that UNC-87 and CLIK-1 have partially redundant function in regulating actin cytoskeletal organization in striated and non-striated muscle cells. However, biochemical studies showed that UNC-87 and CLIK-1 are different in their ability to bundle actin filaments. In this study, I extended comparison between UNC-87 and CLIK-1 and found additional differences in vitro and in vivo. Although UNC-87 and CLIK-1 bound to actin filaments similarly, UNC-87, but not CLIK-1, bound to myosin and inhibited actomyosin ATPase in vitro. In striated muscle, UNC-87 and CLIK-1 were segregated into different subregions within sarcomeric actin filaments. CLIK-1 was concentrated near the actin pointed ends, whereas UNC-87 was enriched toward the actin barbed ends. Restricted localization of UNC-87 was not altered in a clik-1-null mutant, suggesting that their segregated localization is not due to competition between the two related proteins. These results suggest that the two calponin-related proteins have both common and distinct roles in regulating actin filaments.

Mechanical Control of Relaxation using Intact Cardiac Trabeculae.

Diastolic dysfunction is a common phenotype across cardiovascular disease presentations. In addition to elevated cardiac stiffness (elevated left ventricular end-diastolic pressure), impaired cardiac relaxation is a key diagnostic indicator of diastolic dysfunction. While relaxation requires the removal of cytosolic calcium and deactivation of sarcomeric thin filaments, targeting such mechanisms has yet to provide effective treatments. Mechanical mechanisms, such as blood pressure (i.e., afterload), have been theorized to modify relaxation. Recently, we showed that modifying the strain rate of a stretch, not afterload, was both necessary and sufficient to modify the subsequent relaxation rate of myocardial tissue. The strain rate dependence of relaxation, called the mechanical control of relaxation (MCR), can be assessed using intact cardiac trabeculae. This protocol describes the preparation of a small animal model, experimental system and chamber, isolation of the heart and subsequent isolation of a trabecula, preparation of the experimental chamber, and experimental and analysis protocols. Evidence for lengthening strains in the intact heart suggests that MCR might provide new arenas for better characterization of pharmacological treatments, along with a method to assess myofilament kinetics in intact muscles. Therefore, studying the MCR may elucidate a path to novel approaches and new frontiers in the treatment of heart failure.

Modeling mechanisms of the thick-filament off-state dynamics in cardiac muscle.

Cardiac muscle contraction is initiated when sarcomeric thin filaments are activated by a cytosolic Ca2+ transient. Experimental evidence suggests that the contraction strength is modulated by an additional thick-filament activation. Myosin heads in the so-called OFF state are blocked from interacting with thin filaments and therefore cannot produce force. Transitions to the ON state liberate myosin heads, making them available for producing force. Evidence suggests that this transition is itself tension-dependent, but the precise nature and mechanism of this mechanosensitivity remain unclear. The relevance and contribution of passive tension (titin strain) are indicated by some experiments but refuted by others. Furthermore, tension is unlikely to be uniform within the thick filament, which makes the detailed characterization of the mechanosensitivity ambiguous. The precise representation of the mechanosensitivity may have significant repercussions at the tissue and anatomical levels, and hence on cardiac function.

Sarcomeric Thin Filament Associated Cardiomyopathic Mouse Models

Cardiomyopathies are diseases primarily associated with defects in the structure and physiological function of the heart. Hypertrophic and dilated cardiomyopathies are two common conditions associated with severe pathological abnormalities that often lead to heart failure. Studies in the early 1990’s by the Seidman laboratories linked hypertrophic cardiomyopathy with mutations in both thick and thin sarcomeric protein genes. Since then, the development and analysis of animal models of both hypertrophic and dilated cardiomyopathy has significantly advanced our knowledge of the structural, molecular, biochemical, and physiological disease processes that lead to these cardiomyopathic conditions. The focus of this article is an examination of mouse models of hypertrophic and dilated cardiomyopathies with mutations in sarcomeric thin filament protein genes (actin, tropomyosin, troponin T, I, and C) and the information these models provide in our understanding of the disease processes. Special attention addresses the significant role that tropomyosin mutation models have contributed to this information. In addition, we address how various methods have been developed to phenotypically rescue these diseased hearts with respect to their morphological and physiological functions. By thorough analysis of these mouse models, not only can we better understand the disease processes, but there is a great potential for the development of effective therapeutics to treat these severe pathological conditions.

Thin filament cardiomyopathies: A review of genetics, disease mechanisms, and emerging therapeutics.

All muscle contraction occurs due to the cyclical interaction between sarcomeric thin and thick filament proteins within the myocyte. The thin filament consists of the proteins actin, tropomyosin, Troponin C, Troponin I, and Troponin T. Mutations in these proteins can result in various forms of cardiomyopathy, including hypertrophic, restrictive, and dilated phenotypes and account for as many as 30% of all cases of inherited cardiomyopathy. There is significant evidence that thin filament mutations contribute to dysregulation of Ca2+ within the sarcomere and may have a distinct pathomechanism of disease from cardiomyopathy associated with thick filament mutations. A number of distinct clinical findings appear to be correlated with thin-filament mutations: greater degrees of restrictive cardiomyopathy and relatively less left ventricular (LV) hypertrophy and LV outflow tract obstruction than that seen with thick filament mutations, increased morbidity associated with heart failure, increased arrhythmia burden and potentially higher mortality. Most therapies that improve outcomes in heart failure blunt the neurohormonal pathways involved in cardiac remodeling, while most therapies for hypertrophic cardiomyopathy involve use of negative inotropes to reduce LV hypertrophy or septal reduction therapies to reduce LV outflow tract obstruction. None of these therapies directly address the underlying sarcomeric dysfunction associated with thin-filament mutations. With mounting evidence that thin filament cardiomyopathies occur through a distinct mechanism, there is need for therapies targeting the unique, underlying mechanisms tailored for each patient depending on a given mutation.

The Effects of Branched-Chain Amino Acids on the Akt/mTOR Pathway and Nebulin Protein in Joint Fixation-Induced Muscle Atrophy.

It is well known that branched-chain amino acids (BCAAs) promote protein synthesis in skeletal muscle and can cause muscle hypertrophy. However, it has also been reported that they may inhibit muscle atrophy induced by load-bearing and age-related changes. In this study, we investigated the effects of BCAA intake during joint fixation on the levels of protein kinase B (Akt), mammalian target of rapamycin (mTOR), and nebulin in a rat model of joint fixation. Akt and mTOR are signal factors of protein synthesis, whereas nebulin is a structural protein in the muscle. The effects of BCAAs on muscle atrophy were also investigated. The phosphorylation rate of mTOR was higher than that of Akt and increased with BCAA intake in the rat hind limb muscles (soleus) when the ankle joint was fixed. The relative level of nebulin and the phosphorylation rate of Neural Wiskott-Aldrich syndrome protein (N-WASP) also increased as a result of BCAA intake during fixation. This is important because nebulin and N-WASP are involved in the formation of the structure of sarcomere thin filaments. Furthermore, when the cross-sectional areas (CSAs) of different types of muscle fibers were measured during histological evaluation of muscle atrophy, it was found that the inhibitory effect of BCAA on muscle atrophy was higher in Type 1 fibers. Additionally, a positive correlation was found between nebulin level and the CSAs of the muscle fibers. It was found that there is a close relationship between the content of structural proteins and muscle atrophy.

Mutual dependence between tropomodulin and tropomyosin in the regulation of sarcomeric actin assembly in Caenorhabditis elegans striated muscle

Tropomodulin and tropomyosin are important components of sarcomeric thin filaments in striated muscles. Tropomyosin decorates the side of actin filaments and enhances tropomodulin capping at the pointed ends of the filaments. Their functional relationship has been extensively characterized in vitro, but in vivo and cellular studies in mammals are often complicated by the presence of functionally redundant isoforms. Here, we used the nematode Caenorhabditis elegans, which has a relatively simple composition of tropomodulin and tropomyosin genes, and demonstrated that tropomodulin (unc-94) and tropomyosin (lev-11) are mutually dependent on each other in their sarcomere localization and regulation of sarcomeric actin assembly. Mutation of tropomodulin caused sarcomere disorganization with formation of actin aggregates. However, the actin aggregation was suppressed when tropomyosin was depleted in the tropomodulin mutant. Tropomyosin was mislocalized to the actin aggregates in the tropomodulin mutants, while sarcomere localization of tropomodulin was lost when tropomyosin was depleted. These results indicate that tropomodulin and tropomyosin are interdependent in the regulation of organized sarcomeric assembly of actin filaments in vivo.

Muscle ankyrin repeat protein 1 (MARP1) locks titin to the sarcomeric thin filament and is a passive force regulator.

Muscle ankyrin repeat protein 1 (MARP1) is frequently up-regulated in stressed muscle, but its effect on skeletal muscle function is poorly understood. Here, we focused on its interaction with the titin-N2A element, found in titin's molecular spring region. We show that MARP1 binds to F-actin, and that this interaction is stronger when MARP1 forms a complex with titin-N2A. Mechanics and super-resolution microscopy revealed that MARP1 "locks" titin-N2A to the sarcomeric thin filament, causing increased extension of titin's elastic PEVK element and, importantly, increased passive force. In support of this mechanism, removal of thin filaments abolished the effect of MARP1 on passive force. The clinical relevance of this mechanism was established in diaphragm myofibers of mechanically ventilated rats and of critically ill patients. Thus, MARP1 regulates passive force by locking titin to the thin filament. We propose that in stressed muscle, this mechanism protects the sarcomere from mechanical damage.

A Homozygous Deep Intronic Mutation Alters the Splicing of Nebulin Gene in a Patient With Nemaline Myopathy.

Nemaline myopathy is a rare disorder affecting the muscle sarcomere. Mutations in nebulin gene (NEB) are known to be responsible for about 50% of nemaline myopathy cases. Nebulin is a giant protein which is formed integrally with the sarcomeric thin filament. This complex gene is under extensive alternative splicing giving rise to multiple isoforms. In this study, we report a 6-year-old boy presenting with general muscular weaknesses. Identification of rod-shaped structures in the patient' biopsy raised doubt about the presence of a nemaline myopathy. Next-generation sequencing was used to identify a causative mutation for the patient syndrome. A homozygous deep intronic substitution was found in the intron 144 of the NEB. The variant was predicted by in silico tools to create a new donor splice site. Molecular analysis has shown that the mutation could alter splicing events of the nebulin gene leading to a significant decrease of isoforms level. This change in the expression level of nebulin could give rise to functional consequences in the sarcomere. These results are consistent with the phenotypes observed in the patient. Such a discovery of variants in this gene will allow a better understanding of the involvement of nebulin in neuromuscular diseases and help find new treatments for the nemaline myopathy.

The number of Z-repeats and super-repeats in nebulin greatly varies across vertebrates and scales with animal size.

Nebulin is a skeletal muscle protein that associates with the sarcomeric thin filaments and has functions in regulating the length of the thin filament and the structure of the Z-disk. Here we investigated the nebulin gene in 53 species of birds, fish, amphibians, reptiles, and mammals. In all species, nebulin has a similar domain composition that mostly consists of ∼30-residue modules (or simple repeats), each containing an actin-binding site. All species have a large region where simple repeats are organized into seven-module super-repeats, each containing a tropomyosin binding site. The number of super-repeats shows high interspecies variation, ranging from 21 (zebrafish, hummingbird) to 31 (camel, chimpanzee), and, importantly, scales with body size. The higher number of super-repeats in large animals was shown to increase thin filament length, which is expected to increase the sarcomere length for optimal force production, increase the energy efficiency of isometric force production, and lower the shortening velocity of muscle. It has been known since the work of A.V. Hill in 1950 that as species increase in size, the shortening velocity of their muscle is reduced, and the present work shows that nebulin contributes to the mechanistic basis. Finally, we analyzed the differentially spliced simple repeats in nebulin's C terminus, whose inclusion correlates with the width of the Z-disk. The number of Z-repeats greatly varies (from 5 to 18) and correlates with the number of super-repeats. We propose that the resulting increase in the width of the Z-disk in large animals increases the number of contacts between nebulin and structural Z-disk proteins when the Z-disk is stressed for long durations.

CONGENITAL MYOPATHIES 1 – NEMALINE: P.20 Mouse models of nemaline myopathy display structural and functional abnormalities of mitochondria

Nemaline myopathy (NM) is a genetically and clinically heterogenous neuromuscular disorder that can cause death or lifelong disability. Essentially all genes associated with NM are related to the sarcomeric thin filament. Therefore, studies of weakness in NM have focused primarily on issues of skeletal muscle structure and contractility. However, the genetic variability seen does not explain the significant phenotypic heterogeneity observed in NM patients or mouse models, suggesting that additional factors determine disease phenotype.

Nebulin: big protein with big responsibilities

Nebulin, encoded by NEB, is a giant skeletal muscle protein of about 6669 amino acids which forms an integral part of the sarcomeric thin filament. In recent years, the nebula around this protein has been largely lifted resulting in the discovery that nebulin is critical for a number of tasks in skeletal muscle. In this review, we firstly discussed nebulin’s role as a structural component of the thin filament and the Z-disk, regulating the length and the mechanical properties of the thin filament as well as providing stability to myofibrils by interacting with structural proteins within the Z-disk. Secondly, we reviewed nebulin’s involvement in the regulation of muscle contraction, cross-bridge cycling kinetics, Ca2+-homeostasis and excitation contraction (EC) coupling. While its role in Ca2+-homeostasis and EC coupling is still poorly understood, a large number of studies have helped to improve our knowledge on how nebulin affects skeletal muscle contractile mechanics. These studies suggest that nebulin affects the number of force generating actin-myosin cross-bridges and may also affect the force that each cross-bridge produces. It may exert this effect by interacting directly with actin and myosin and/or indirectly by potentially changing the localisation and function of the regulatory complex (troponin and tropomyosin). Besides unravelling the biology of nebulin, these studies are particularly helpful in understanding the patho-mechanism of myopathies caused by NEB mutations, providing knowledge which constitutes the critical first step towards the development of therapeutic interventions. Currently, effective treatments are not available, although a number of therapeutic strategies are being investigated.

Assembly and Maintenance of Sarcomere Thin Filaments and Associated Diseases.

Sarcomere assembly and maintenance are essential physiological processes required for cardiac and skeletal muscle function and organism mobility. Over decades of research, components of the sarcomere and factors involved in the formation and maintenance of this contractile unit have been identified. Although we have a general understanding of sarcomere assembly and maintenance, much less is known about the development of the thin filaments and associated factors within the sarcomere. In the last decade, advancements in medical intervention and genome sequencing have uncovered patients with novel mutations in sarcomere thin filaments. Pairing this sequencing with reverse genetics and the ability to generate patient avatars in model organisms has begun to deepen our understanding of sarcomere thin filament development. In this review, we provide a summary of recent findings regarding sarcomere assembly, maintenance, and disease with respect to thin filaments, building on the previous knowledge in the field. We highlight debated and unknown areas within these processes to clearly define open research questions.

The Troponin-I fast skeletal muscle is reliable marker for the determination of vitality in the suicide hanging

Troponin I (TnI) is the inhibitory subunit of the troponin complex in the sarcomeric thin filament of striated muscle and plays a central role in the calcium regulation of contraction and relaxation. Vertebrate TnI has evolved into three isoforms encoded by three homologous genes: TNNI1 for slow skeletal muscle TnI, TNNI2 for fast skeletal muscle TnI and TNNI3 for cardiac TnI, which are expressed under muscle type-specific and developmental regulations in both the atrium and ventricle of the heart. Skeletal muscle TnI (both sTnI iso-forms) have been proposed as a sensitive and fast fiber-specific serum marker of skeletal muscle damage; fsTnI concentration in increased peripheral blood when fast twitch fibers were damaged. In our study we investigate if the ‘Troponin I, fast skeletal muscle’ can also be used as a reliable diagnostic tool in forensic practice, to perform differential diagnosis about vitality in suicide by hanging and simulated hanging (suspension of the victim after murder). We selected 8 women and 13 men, mean age 52.2 years, who died from suicidal hanging. The ligature material used for hanging was soft material in 11 cases and hard material in 10 cases. We chose cases as a control group of adults (n = 10; six women, four men, mean age 47.3 years) that died from opioid overdose (n = 2), car accident (n = 3) and sudden cardiac death (n = 5). Those deaths were characterized by their rapidity. To test the Anti-Troponin I fast skeletal muscle Antibody (Abcam clone-134,838), we used a case of a subject who died of myocardial infarction (timing infarct dated to 24–36 h prior to death). The reactions to Troponin I (namely the amount and extent of marker depletion) was scored for each section from 0 to −3: 0 = no loss of staining; −1 = minimal decrease in staining, compared to normally stained tissue; −2 = clear decrease in staining with some positivity (brown color) remaining; and −3 = no positive (brown) staining. The set of results obtained leads us to believe that the use of this antibody (Anti-Troponin I fast skeletal muscle antibody) is very promising to be able to make a certain differential diagnosis between antemortem and postmortem hangings. It should be emphasized that the present study seems to open new and promising horizons in the possibility to discriminate between suicidal hanging and simulated hanging (suspension of the victim after murder).

CapZ integrates several signaling pathways in response to mechanical stiffness.

Muscle adaptation is a response to physiological demand elicited by changes in mechanical load, hormones, or metabolic stress. Cytoskeletal remodeling processes in many cell types are thought to be primarily regulated by thin filament formation due to actin-binding accessory proteins, such as the actin-capping protein. Here, we hypothesize that in muscle, the actin-capping protein (named CapZ) integrates signaling by a variety of pathways, including phosphorylation and phosphatidylinositol 4,5-bisphosphate (PIP2) binding, to regulate muscle fiber growth in response to mechanical load. To test this hypothesis, we assess mechanotransduction signaling that regulates muscle growth using neonatal rat ventricular myocytes cultured on substrates with the stiffness of the healthy myocardium (10 kPa), fibrotic myocardium (100 kPa), or glass. We investigate how PIP2 signaling affects CapZ using the PIP2 sequestering agent neomycin and the effect of PKC-mediated CapZ phosphorylation using the PKC-activating drug phorbol 12-myristate 13-acetate (PMA). Molecular simulations suggest that close interactions between PIP2 and the β-tentacle of CapZ are modified by phosphorylation at T267. Fluorescence recovery after photobleaching (FRAP) demonstrates that the kinetic binding constant of CapZ to sarcomeric thin filaments in living muscle cells increases with stiffness or PMA treatment but is diminished by PIP2 reduction. Furthermore, CapZ with a deletion of the β-tentacle that lacks the phosphorylation site T267 shows increased FRAP kinetics with lack of sensitivity to PMA treatment or PIP2 reduction. Förster resonance energy transfer (FRET) probes the molecular interactions between PIP2 and CapZ, which are decreased by PIP2 availability or by the β-tentacle truncation. These data suggest that CapZ is bound to actin tightly in the idle, locked state, with little phosphorylation or PIP2 binding. However, this tight binding is loosened in growth states triggered by mechanical stimuli such as substrate stiffness, which may have relevance to fibrotic heart disease.

Abstract 17079: MYBPC3 Truncation Mutations Cause Contractile Dysregulation in iPSC-Derived Cardiomyocytes

The mechanism of MYBPC3 (encoding cardiac myosin binding protein C, MyBP-C) truncation mutations, the most common genetic cause of hypertrophic cardiomyopathy, has been incompletely resolved. We hypothesized that truncating MYBPC3 mutations cause myofibrillar protein assembly defects and/or contractile dysfunction. Methods and Results: Control iPSCs were CRISPR/Cas9-edited to create cell lines with homozygous (homCT) and heterozygous (hetCT) C-terminal MYBPC3 truncation mutations and heterozygous MYBPC3 promoter knock-out (hetPROM). HetCT was further edited to add an N-terminal flag tag (FhetCT). Purified cardiomyocytes were assayed at day ~25. RNAseq showed a 50% reduction in MYBPC3 mRNA in hetCT (p<0.01). Full ablation of MyBP-C was demonstrated in homCT but MyBP-C content was not reduced in hetCT or hetPROM by quantitative mass spectrometry. Immunofluorescence and co-IP of FhetCT showed no truncated MyBP-C. Sarcomere assembly, quantified by aligned myofilament number after replating onto 7:1 rectangular micropatterns, did not differ between lines; homCT (23±3), hetCT (23±3), hetPROM (22±3 myofilaments), control (22±3), p=NS. Quantitative mass spectrometry demonstrated that the stoichiometry of other major thick and thin filament proteins was not altered (all p=NS). Maximum force was reduced in homCT (866±366 uN, p<0.001) and hetCT (1213±545 uN, p=0.03) vs controls (1509±441 uN), and not significantly different for hetPROM (1238±360 uN, p=0.07), when measured in single micropatterned iPSC-CMs on 8.7 kPa hydrogels by traction force microscopy. Time to peak contraction was shorter in homCT (0.27 s, p=0.02) vs control (0.42 sec), but not in hetCT (0.46 s) and hetPROM (0.49 s). Contractile deceleration time was reduced only in homCT (0.12 s vs 0.25 s, p=0.002). Conclusions: Heterozygous MYBPC3 truncation mutations result in haploinsufficent mRNA with steady state compensation of MYBP3 protein and no MYBPC3 truncated peptide. Even complete ablation of MyBP-C loss does not alter the overall stoichiometry of other sarcomeric thick and thin filament proteins. MyBP-C ablation and heterozygous truncating mutations impair maximal contractile force, strongly implicating contractile dysregulation as the primary mechanism of MYBPC3-HCM.

CONGENITAL MYOPATHIES: GENERAL AND RYR1: P.48Hypercontractile congenital muscle stiffness

Tropomyosin 3 encoded by the TPM3 gene is a member of the acting binding tropomyosin family, a component of the sarcomeric thin filaments troponin/ tropomyosin complex that is essential in muscle contraction by regulating the calcium dependent binding of the myosin head to the actin filament. Mutations in TPM3 cause a clinical and histopathological heterogeneous group of neuromuscular disorders characterized by congenital hypotonia and weakness that includes cap myopathy, congenital fiber type disproportion and nemaline myopathy. Recently, a new phenotype characterized by hypercontractile stats and stiffness was described, most probably due to mutations that increase calcium sensitivity of the troponin-tropomyosin complex, resulting in excessively sensitized excitation-contraction coupling of the contractile apparatus. This study presents the case of a one-year-old female, daughter of non-consanguineous parents, of an uneventful gestation, was born full term with Apgar 3/8 and presented generalized hypertonia and hip dysplasia from the first month of life. She had no cephalic support and was not able to sit without support because of the stiffness. Apparently, she has normal cognition. She has a short neck, hypertrophy of the cervical, paravertebral and proximal muscles of the upper limbs and a bell-shaped chest. During the neurological exam, she was able to move against gravity with all four limbs, but the strength scale evaluation was impaired due to by the stiffness. Deeper tendon reflexes, sensation and cranial nerves were normal. The first diagnosis was Schwartz Jampel syndrome; however, myotonic discharges were not detected in the electromyography exam. Computed tomography of the brain, pelvic girdle radiography, serum CK level, and mucopolysaccharidosis screen were normal. Echocardiogram showed patent foramen oval with left-right flow. Ultrasound of the hip joint showed dysplasia on the left side. Clinical, phenotypic and pathophysiological spectrum of TPM3 mutations are broader than once thought, and now joins congenital hypercontractile and stiffness phenotype.

Dysfunctional sarcomere contractility contributes to muscle weakness in ACTA1-related nemaline myopathy (NEM3).

Nemaline myopathy (NM) is one of the most common congenital nondystrophic myopathies and is characterized by muscle weakness, often from birth. Mutations in ACTA1 are a frequent cause of NM (ie, NEM3). ACTA1 encodes alpha-actin 1, the main constituent of the sarcomeric thin filament. The mechanisms by which mutations in ACTA1 contribute to muscle weakness in NEM3 are incompletely understood. We hypothesized that sarcomeric dysfunction contributes to muscle weakness in NEM3 patients. To test this hypothesis, we performed contractility measurements in individual muscle fibers and myofibrils obtained from muscle biopsies of 14 NEM3 patients with different ACTA1 mutations. To identify the structural basis for impaired contractility, low angle X-ray diffraction and stimulated emission-depletion microscopy were applied. Our findings reveal that muscle fibers of NEM3 patients display a reduced maximal force-generating capacity, which is caused by dysfunctional sarcomere contractility in the majority of patients, as revealed by contractility measurements in myofibrils. Low angle X-ray diffraction and stimulated emission-depletion microscopy indicate that dysfunctional sarcomere contractility in NEM3 patients involves a lower number of myosin heads binding to actin during muscle activation. This lower number is not the result of reduced thin filament length. Interestingly, the calcium sensitivity of force is unaffected in some patients, but decreased in others. Dysfunctional sarcomere contractility is an important contributor to muscle weakness in the majority of NEM3 patients. This information is crucial for patient stratification in future clinical trials. Ann Neurol 2018;83:269-282.

ACTA1-Related Nemaline Myopathy Mutations Engender a Range of Structural and Functional Phenotypes in Drosophila Indirect Flight Muscles

Nemaline myopathy is a congenital disorder characterized by muscle weakness. Dominant-negative disease-causing mutations have been identified in various genes that encode sarcomeric thin filament proteins. of these, mutations in skeletal muscle α-actin (ACTA1) account for ∼25% of all reported cases. To investigate the myopathic phenotypes from the level of whole muscle down through the level of single molecules, we generated multiple transgenic Drosophila fly lines expressing three different mutations - V37L, D288G and F352S - in Act88F, the indirect flight muscle (IFM) actin gene. A gradation in flight behavior was observed (V37L>D288G>F352S), which correlated with the observed differences in muscle fiber morphology. While most of these mutations depress muscle function in patients, the F352S lesion elevates myosin cross-bridge strain and steady-state isometric force production and, thereby, apparently increases contractile function. Confocal and electron microscopy revealed breaks in mutant F352S IFM fibers and extensive myofibrillar and myofilamentous disarray. Z-lines showed streaming and formed “zebra bodies”. The microscopic alterations are consistent with excessive acto-myosin interactions, unevenly distributed force, and destructive hypercontraction. Interestingly, in vitro sliding velocities of purified IFM F-actin from wild-type (3.17±0.12 μm/s) vs. F352S mutant heterozygous (2.88±0.09 μm/s, mean±SEM) flies did not significantly differ. Similarly, no differences in maximum calcium-activated velocity (3.88±0.11 vs. 3.77±0.10 μm/s) or in cooperativity (nH = 2.13±0.27 vs. 2.62±0.46) were observed for F-actin in the presence of vertebrate tropomyosin and troponin. However, relative to control, F352S heterozygous filaments showed a significant (p<0.01) increase in calcium sensitivity (pCa50 = 5.99±0.03 vs. 6.11±0.03), consistent with a mutation-induced gain in molecular function. Going forward, our novel Drosophila models will be used to screen for in vivo modifiers of mutant muscle performance. Supported by NIH R01HL124091 to AC.