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Background: Nemaline myopathy type 6 (NEM6) or KBTBD13-related congenital myopathy is the most prevalent type of nemaline myopathy in the Netherlands and is characterised by mild childhood-onset axial, proximal and distal muscle weakness with prominent neck flexor weakness combined with slowness of movements. The most prevalent variant in the Netherlands is the c.1222C > T p.(Arg408Cys) variant in the KBTBD13 gene, also called the Dutch founder variant., Objective: To provide a comprehensive clinical and functional characterisation of three patients to assess the pathogenicity of a newly identified variant in the KBTBD13 gene., Results: We present three cases (Patient 1: female, 76 years old; Patient 2: male, 63 years old; and his brother Patient 3: male, 61 years old) with a c.1222C > A p.(Arg408Ser) variant in the KBTBD13 gene. Patient 1 was also included previously in a histopathological study on NEM6. Symptoms of muscle weakness started in childhood and progressed to impaired functional abilities in adulthood. All three patients reported slowness of movements. On examination, they have mild axial, proximal and distal muscle weakness. None of the patients exhibited cardiac abnormalities. Spirometry in two patients showed a restrictive lung pattern. Muscle ultrasound showed symmetrically increased echogenicity indicating fatty replacement and fibrosis in a subset of muscles and histopathological analyses revealed nemaline rods and cores. Slower muscle relaxation kinetics with in vivo functional tests was observed. This was confirmed by in vitro functional tests showing impaired relaxation kinetics in isolated muscle fibres. We found a genealogic link between patient 1, and patient 2 and 3 nine generations earlier., Conclusions: The c.1222C > A p.(Arg408Ser) variant in the KBTBD13 gene is a likely pathogenic variant causing NEM6.

Troponin I (TnI) regulates thin filament activation and muscle contraction. Two isoforms, TnI-fast ( TNNI2 ) and TnI-slow ( TNNI1 ), are predominantly expressed in fast- and slow-twitch myofibers, respectively. TNNI2 variants are a rare cause of arthrogryposis, whereas TNNI1 variants have not been conclusively established to cause skeletal myopathy. We identified recessive loss-of-function TNNI1 variants as well as dominant gain-of-function TNNI1 variants as a cause of muscle disease, each with distinct physiological consequences and disease mechanisms. We identified three families with biallelic TNNI1 variants (F1: p.R14H/c.190-9G>A, F2 and F3: homozygous p.R14C), resulting in loss of function, manifesting with early-onset progressive muscle weakness and rod formation on histology. We also identified two families with a dominantly acting heterozygous TNNI1 variant (F4: p.R174Q and F5: p.K176del), resulting in gain of function, manifesting with muscle cramping, myalgias, and rod formation in F5. In zebrafish, TnI proteins with either of the missense variants (p.R14H; p.R174Q) incorporated into thin filaments. Molecular dynamics simulations suggested that the loss-of-function p.R14H variant decouples TnI from TnC, which was supported by functional studies showing a reduced force response of sarcomeres to submaximal [Ca 2+ ] in patient myofibers. This contractile deficit could be reversed by a slow skeletal muscle troponin activator. In contrast, patient myofibers with the gain-of-function p.R174Q variant showed an increased force to submaximal [Ca 2+ ], which was reversed by the small-molecule drug mavacamten. Our findings demonstrated that TNNI1 variants can cause muscle disease with variant-specific pathomechanisms, manifesting as either a hypo- or a hypercontractile phenotype, suggesting rational therapeutic strategies for each mechanism.

Nemaline myopathies are the most common form of congenital myopathies. Variants in ACTA1 (NEM3) comprise 15-25% of all nemaline myopathy cases. Patients harboring variants in ACTA1 present with a heterogeneous disease course characterized by stable or progressive muscle weakness and, in severe cases, respiratory failure and death. To date, no specific treatments are available. Since NEM3 is an actin-based thin filament disease, we tested the ability of tirasemtiv, a fast skeletal muscle troponin activator, to improve skeletal muscle function in a mouse model of NEM3, harboring the patient-based p.Asp286Gly variant in Acta1. Acute and long-term tirasemtiv treatment significantly increased muscle contractile capacity at submaximal stimulation frequencies in both fast-twitch extensor digitorum longus and gastrocnemius muscle, and intermediate-twitch diaphragm muscle in vitro and in vivo. Additionally, long-term tirasemtiv treatment in NEM3 mice resulted in a decreased respiratory rate with preserved minute volume, suggesting more efficient respiration. Altogether, our data support the therapeutic potential of fast skeletal muscle troponin activators in alleviating skeletal muscle weakness in a mouse model of NEM3 caused by the Acta1:p.Asp286Gly variant., (© 2024 Galli et al.)

Muscle weakness is a hallmark of inherited or acquired myopathies. It is a major cause of functional impairment and can advance to life-threatening respiratory insufficiency. During the past decade, several small-molecule drugs that improve the contractility of skeletal muscle fibers have been developed. In this review, we provide an overview of the available literature and the mechanisms of action of small-molecule drugs that modulate the contractility of sarcomeres, the smallest contractile units in striated muscle, by acting on myosin and troponin. We also discuss their use in the treatment of skeletal myopathies. The first of three classes of drugs discussed here increase contractility by decreasing the dissociation rate of calcium from troponin and thereby sensitizing the muscle to calcium. The second two classes of drugs directly act on myosin and stimulate or inhibit the kinetics of myosin-actin interactions, which may be useful in patients with muscle weakness or stiffness. NEW & NOTEWORTHY During the past decade, several small molecule drugs that improve the contractility of skeletal muscle fibers have been developed. In this review, we provide an overview of the available literature and the mechanisms of action of small molecule drugs that modulate the contractility of sarcomeres, the smallest contractile units in striated muscle, by acting on myosin and troponin.

KBTBD13 variants cause nemaline myopathy type 6 (NEM6). The majority of NEM6 patients harbors the Dutch founder variant, c.1222C>T, p.Arg408Cys (KBTBD13 p.R408C). Although KBTBD13 is expressed in cardiac muscle, cardiac involvement in NEM6 is unknown. Here, we constructed pedigrees of three families with the KBTBD13 p.R408C variant. In 65 evaluated patients, 12% presented with left ventricle dilatation, 29% with left ventricular ejection fraction< 50%, 8% with atrial fibrillation, 9% with ventricular tachycardia, and 20% with repolarization abnormalities. Five patients received an implantable cardioverter defibrillator, three cases of sudden cardiac death were reported. Linkage analysis confirmed cosegregation of the KBTBD13 p.R408C variant with the cardiac phenotype. Mouse studies revealed that (1) mice harboring the Kbtbd13 p.R408C variant display mild diastolic dysfunction; (2) Kbtbd13-deficient mice have systolic dysfunction. Hence, (1) KBTBD13 is associated with cardiac dysfunction and cardiomyopathy; (2) KBTBD13 should be added to the cardiomyopathy gene panel; (3) NEM6 patients should be referred to the cardiologist., (© 2022 The Authors. Human Mutation published by Wiley Periodicals LLC.)

Assessing the umami taste of seaweed on a chemical level can inform the use and selection of seaweed in European cuisine. Accordingly, we developed a method for the simultaneous extraction, separate clean-up and analysis of 21 free amino acids and 10 free nucleotides by reversed phase and mixed-mode HPLC respectively. Of multiple mouth emulating solvents, extracting in Milli-Q at 35 °C was found most suitable. This method showed good linearity (R 2  > 0.9996), resolution (R s  ≥ 1.5) and picomole detection limits, and was successfully applied to determine the Equivalent Umami Concentration (EUC) and Taste Activity Values (TAV) of seven Dutch seaweed species. Phaeophyceae showed the highest EUC, followed by Chlorophyceae and Rhodophyceae (≈ 9.5, 3.7 and 1.1 g/100 g respectively). Glutamic acid always exceeded the TAV, while other umami compounds were species specific. Our method can accurately predict umami intensity and therefore contributes towards species selection for the European palette., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Nemaline myopathy, a disease of the actin-based thin filament, is one of the most frequent congenital myopathies. To date, no specific therapy is available to treat muscle weakness in nemaline myopathy. We tested the ability of tirasemtiv, a fast skeletal troponin activator that targets the thin filament, to augment muscle force-both in vivo and in vitro-in a nemaline myopathy mouse model with a mutation (H40Y) in Acta1. In Acta1H40Y mice, treatment with tirasemtiv increased the force response of muscles to submaximal stimulation frequencies. This resulted in a reduced energetic cost of force generation, which increases the force production during a fatigue protocol. The inotropic effects of tirasemtiv were present in locomotor muscles and, albeit to a lesser extent, in respiratory muscles, and they persisted during chronic treatment, an important finding as respiratory failure is the main cause of death in patients with congenital myopathy. Finally, translational studies on permeabilized muscle fibers isolated from a biopsy of a patient with the ACTA1H40Y mutation revealed that at physiological Ca2+ concentrations, tirasemtiv increased force generation to values that were close to those generated in muscle fibers of healthy subjects. These findings indicate the therapeutic potential of fast skeletal muscle troponin activators to improve muscle function in nemaline myopathy due to the ACTA1H40Y mutation, and future studies should assess their merit for other forms of nemaline myopathy and for other congenital myopathies., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Troponin C (TnC) is a critical regulator of skeletal muscle contraction; it binds Ca2+ to activate muscle contraction. Surprisingly, the gene encoding fast skeletal TnC (TNNC2) has not yet been implicated in muscle disease. Here, we report 2 families with pathogenic variants in TNNC2. Patients present with a distinct, dominantly inherited congenital muscle disease. Molecular dynamics simulations suggested that the pathomechanisms by which the variants cause muscle disease include disruption of the binding sites for Ca2+ and for troponin I. In line with these findings, physiological studies in myofibers isolated from patients' biopsies revealed a markedly reduced force response of the sarcomeres to [Ca2+]. This pathomechanism was further confirmed in experiments in which contractile dysfunction was evoked by replacing TnC in myofibers from healthy control subjects with recombinant, mutant TnC. Conversely, the contractile dysfunction of myofibers from patients was repaired by replacing endogenous, mutant TnC with recombinant, wild-type TnC. Finally, we tested the therapeutic potential of the fast skeletal muscle troponin activator tirasemtiv in patients' myofibers and showed that the contractile dysfunction was repaired. Thus, our data reveal that pathogenic variants in TNNC2 cause congenital muscle disease, and they provide therapeutic angles to repair muscle contractility.

Nemaline myopathy type 6 (NEM6), KBTBD13-related congenital myopathy is caused by mutated KBTBD13 protein that interacts improperly with thin filaments/actin, provoking impaired muscle-relaxation kinetics. We describe muscle morphology in 18 Dutch NEM6 patients and correlate it with clinical phenotype and pathophysiological mechanisms. Rods were found in in 85% of biopsies by light microscopy, and 89% by electron microscopy. A peculiar ring disposition of rods resulting in ring-rods fiber was observed. Cores were found in 79% of NEM6 biopsies by light microscopy, and 83% by electron microscopy. Electron microscopy also disclosed granulofilamentous protein material in 9 biopsies. Fiber type 1 predominance and prominent nuclear internalization were found. Rods were immunoreactive for α-actinin and myotilin. Areas surrounding the rods showed titin overexpression suggesting derangement of the surrounding sarcomeres. NEM6 myopathology hallmarks are prominent cores, rods including ring-rods fibers, nuclear clumps, and granulofilamentous protein material. This material might represent the histopathologic epiphenomenon of altered interaction between mutated KBTBD13 protein and thin filaments. We claim to classify KBTBD13-related congenital myopathy as rod-core myopathy., (© 2021 American Association of Neuropathologists, Inc. All rights reserved.)

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-Pérot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

The mechanisms that modulate the kinetics of muscle relaxation are critically important for muscle function. A prime example of the impact of impaired relaxation kinetics is nemaline myopathy caused by mutations in KBTBD13 (NEM6). In addition to weakness, NEM6 patients have slow muscle relaxation, compromising contractility and daily life activities. The role of KBTBD13 in muscle is unknown, and the pathomechanism underlying NEM6 is undetermined. A combination of transcranial magnetic stimulation-induced muscle relaxation, muscle fiber- and sarcomere-contractility assays, low-angle x-ray diffraction, and superresolution microscopy revealed that the impaired muscle-relaxation kinetics in NEM6 patients are caused by structural changes in the thin filament, a sarcomeric microstructure. Using homology modeling and binding and contractility assays with recombinant KBTBD13, Kbtbd13-knockout and Kbtbd13R408C-knockin mouse models, and a GFP-labeled Kbtbd13-transgenic zebrafish model, we discovered that KBTBD13 binds to actin - a major constituent of the thin filament - and that mutations in KBTBD13 cause structural changes impairing muscle-relaxation kinetics. We propose that this actin-based impaired relaxation is central to NEM6 pathology.

Myopathies due to recessive MYH7 mutations are exceedingly rare, reported in only two families to date. We describe three patients from two families (from Australia and the UK) with a myopathy caused by recessive mutations in MYH7. The Australian family was homozygous for a c.5134C > T, p.Arg1712Trp mutation, whilst the UK patient was compound heterozygous for a truncating (c.4699C > T; p.Gln1567*) and a missense variant (c.4664A > G; p.Glu1555Gly). All three patients shared key clinical features, including infancy/childhood onset, pronounced axial/proximal weakness, spinal rigidity, severe scoliosis, and normal cardiac function. There was progressive respiratory impairment necessitating non-invasive ventilation despite preserved ambulation, a combination of features often seen in SEPN1- or NEB-related myopathies. On biopsy, the Australian proband showed classical myosin storage myopathy features, while the UK patient showed multi-minicore like areas. To establish pathogenicity of the Arg1712Trp mutation, we expressed mutant MYH7 protein in COS-7 cells, observing abnormal mutant myosin aggregation compared to wild-type. We describe skinned myofiber studies of patient muscle and hypertrophy of type II myofibers, which may be a compensatory mechanism. In summary, we have expanded the phenotype of ultra-rare recessive MYH7 disease, and provide novel insights into associated changes in muscle physiology., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: 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., Methods: 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., Results: 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., Interpretation: 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., (© 2018 American Neurological Association.)

Nemaline myopathy (NM) is among the most common non-dystrophic congenital myopathies (incidence 1:50.000). Hallmark features of NM are skeletal muscle weakness and the presence of nemaline bodies in the muscle fiber. The clinical phenotype of NM patients is quite diverse, ranging from neonatal death to normal lifespan with almost normal motor function. As the respiratory muscles are involved as well, severely affected patients are ventilator-dependent. The mechanisms underlying muscle weakness in NM are currently poorly understood. Therefore, no therapeutic treatment is available yet.Eleven implicated genes have been identified: ten genes encode proteins that are either components of thin filament, or are thought to contribute to stability or turnover of thin filament proteins. The thin filament is a major constituent of the sarcomere, the smallest contractile unit in muscle. It is at this level of contraction - thin-thick filament interaction - where muscle weakness originates in NM patients.This review focusses on how sarcomeric gene mutations directly compromise sarcomere function in NM. Insight into the contribution of sarcomeric dysfunction to muscle weakness in NM, across the genes involved, will direct towards the development of targeted therapeutic strategies.

Nemaline myopathy is among the most common non-dystrophic congenital myopathies, and is characterized by the presence of nemaline rods in skeletal muscles fibers, general muscle weakness, and hypotonia. Although respiratory failure is the main cause of death in nemaline myopathy, only little is known regarding the contractile strength of the diaphragm, the main muscle of inspiration. To investigate diaphragm contractility, in the present study we took advantage of a mouse model for nebulin-based nemaline myopathy that we recently developed. In this mouse model, exon 55 of Neb is deleted (Neb ΔExon55 ), a mutation frequently found in patients. Diaphragm contractility was determined in permeabilized muscle fibers and was compared to the contractility of permeabilized fibers from three peripheral skeletal muscles: soleus, extensor digitorum longus, and gastrocnemius. The force generating capacity of diaphragm muscle fibers of Neb ΔExon55 mice was reduced to 25% of wildtype levels, indicating severe contractile weakness. The contractile weakness of diaphragm fibers was more pronounced than that observed in soleus muscle, but not more pronounced than that observed in extensor digitorum longus and gastrocnemius muscles. The reduced muscle contractility was at least partly caused by changes in cross-bridge cycling kinetics which reduced the number of bound cross-bridges. The severe diaphragm weakness likely contributes to the development of respiratory failure in Neb ΔExon55 mice and might explain their early, postnatal death., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objective: Mutations in TPM3, encoding Tpm3.12, cause a clinically and histopathologically diverse group of myopathies characterized by muscle weakness. We report two patients with novel de novo Tpm3.12 single glutamic acid deletions at positions ΔE218 and ΔE224, resulting in a significant hypercontractile phenotype with congenital muscle stiffness, rather than weakness, and respiratory failure in one patient., Methods: The effect of the Tpm3.12 deletions on the contractile properties in dissected patient myofibers was measured. We used quantitative in vitro motility assay to measure Ca(2+) sensitivity of thin filaments reconstituted with recombinant Tpm3.12 ΔE218 and ΔE224., Results: Contractility studies on permeabilized myofibers demonstrated reduced maximal active tension from both patients with increased Ca(2+) sensitivity and altered cross-bridge cycling kinetics in ΔE224 fibers. In vitro motility studies showed a two-fold increase in Ca(2+) sensitivity of the fraction of filaments motile and the filament sliding velocity concentrations for both mutations., Interpretation: These data indicate that Tpm3.12 deletions ΔE218 and ΔE224 result in increased Ca(2+) sensitivity of the troponin-tropomyosin complex, resulting in abnormally active interaction of the actin and myosin complex. Both mutations are located in the charged motifs of the actin-binding residues of tropomyosin 3, thus disrupting the electrostatic interactions that facilitate accurate tropomyosin binding with actin necessary to prevent the on-state. The mutations destabilize the off-state and result in excessively sensitized excitation-contraction coupling of the contractile apparatus. This work expands the phenotypic spectrum of TPM3-related disease and provides insights into the pathophysiological mechanisms of the actin-tropomyosin complex., (© 2015 American Neurological Association.)

Dominant mutations in TPM3, encoding α-tropomyosinslow, cause a congenital myopathy characterized by generalized muscle weakness. Here, we used a multidisciplinary approach to investigate the mechanism of muscle dysfunction in 12 TPM3-myopathy patients. We confirm that slow myofibre hypotrophy is a diagnostic hallmark of TPM3-myopathy, and is commonly accompanied by skewing of fibre-type ratios (either slow or fast fibre predominance). Patient muscle contained normal ratios of the three tropomyosin isoforms and normal fibre-type expression of myosins and troponins. Using 2D-PAGE, we demonstrate that mutant α-tropomyosinslow was expressed, suggesting muscle dysfunction is due to a dominant-negative effect of mutant protein on muscle contraction. Molecular modelling suggested mutant α-tropomyosinslow likely impacts actin-tropomyosin interactions and, indeed, co-sedimentation assays showed reduced binding of mutant α-tropomyosinslow (R168C) to filamentous actin. Single fibre contractility studies of patient myofibres revealed marked slow myofibre specific abnormalities. At saturating [Ca(2+)] (pCa 4.5), patient slow fibres produced only 63% of the contractile force produced in control slow fibres and had reduced acto-myosin cross-bridge cycling kinetics. Importantly, due to reduced Ca(2+)-sensitivity, at sub-saturating [Ca(2+)] (pCa 6, levels typically released during in vivo contraction) patient slow fibres produced only 26% of the force generated by control slow fibres. Thus, weakness in TPM3-myopathy patients can be directly attributed to reduced slow fibre force at physiological [Ca(2+)], and impaired acto-myosin cross-bridge cycling kinetics. Fast myofibres are spared; however, they appear to be unable to compensate for slow fibre dysfunction. Abnormal Ca(2+)-sensitivity in TPM3-myopathy patients suggests Ca(2+)-sensitizing drugs may represent a useful treatment for this condition., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is characterized by generalized skeletal muscle weakness, often from birth. To date, no therapy exists that enhances the contractile strength of muscles of NM patients. Mutations in NEB, encoding the giant protein nebulin, are the most common cause of NM. The pathophysiology of muscle weakness in NM patients with NEB mutations (NEB-NM) includes a lower calcium-sensitivity of force generation. We propose that the lower calcium-sensitivity of force generation in NEB-NM offers a therapeutic target. Levosimendan is a calcium sensitizer that is approved for use in humans and has been developed to target cardiac muscle fibers. It exerts its effect through binding to slow skeletal/cardiac troponin C. As slow skeletal/cardiac troponin C is also the dominant troponin C isoform in slow-twitch skeletal muscle fibers, we hypothesized that levosimendan improves slow-twitch muscle fiber strength at submaximal levels of activation in patients with NEB-NM., Methods: To test whether levosimendan affects force production, permeabilized slow-twitch muscle fibers isolated from biopsies of NEB-NM patients and controls were exposed to levosimendan and the force response was measured., Results: No effect of levosimendan on muscle fiber force in NEB-NM and control skeletal muscle fibers was found, both at a submaximal calcium level using incremental levosimendan concentrations, and at incremental calcium concentrations in the presence of levosimendan. In contrast, levosimendan did significantly increase the calcium-sensitivity of force in human single cardiomyocytes. Protein analysis confirmed that the slow skeletal/cardiac troponin C isoform was present in the skeletal muscle fibers tested., Conclusions: These findings indicate that levosimendan does not improve the contractility in human skeletal muscle fibers, and do not provide rationale for using levosimendan as a therapeutic to restore muscle weakness in NEB-NM patients. We stress the importance of searching for compounds that improve the calcium-sensitivity of force generation of slow-twitch muscle fibers. Such compounds provide an appealing approach to restore muscle force in patients with NEB-NM, and also in patients with other neuromuscular disorders.

Nemaline myopathy (NM) is a rare congenital myopathy characterised by hypotonia, muscle weakness, and often skeletal muscle deformities with the presence of nemaline bodies (rods) in the muscle biopsy. The nebulin (NEB) gene is the most commonly mutated and is thought to account for approximately 50% of genetically diagnosed cases of NM. We undertook a detailed muscle morphological analysis of 14 NEB-mutated NM patients with different clinical forms to define muscle pathological patterns and correlate them with clinical course and genotype. Three groups were identified according to clinical severity. Group 1 (n = 5) comprises severe/lethal NM and biopsy in the first days of life. Group 2 (n = 4) includes intermediate NM and biopsy in infancy. Group 3 (n = 5) comprises typical/mild NM and biopsy in childhood or early adult life. Biopsies underwent histoenzymological, immunohistochemical and ultrastructural analysis. Fibre type distribution patterns, rod characteristics, distribution and localization were investigated. Contractile performance was studied in muscle fibre preparations isolated from seven muscle biopsies from each of the three groups. G1 showed significant myofibrillar dissociation and smallness with scattered globular rods in one third of fibres; there was no type 1 predominance. G2 presented milder sarcomeric dissociation, dispersed or clustered nemaline bodies, and type 1 predominance/uniformity. In contrast, G3 had well-delimited clusters of subsarcolemmal elongated rods and type 1 uniformity without sarcomeric alterations. In accordance with the clinical and morphological data, functional studies revealed markedly low forces in muscle bundles from G1 and a better contractile performance in muscle bundles from biopsies of patients from G2, and G3.In conclusion NEB-mutated NM patients present a wide spectrum of morphological features. It is difficult to establish firm genotype phenotype correlation. Interestingly, there was a correlation between clinical severity on the one hand and the degree of sarcomeric dissociation and contractility efficiency on the other. By contrast the percentage of fibres occupied by rods, as well as the quantity and the sub sarcolemmal position of rods, appears to inversely correlate with severity. Based on our observations, we propose myofibrillar dissociation and changes in contractility as an important cause of muscle weakness in NEB-mutated NM patients.

Background: Nemaline myopathy-the most common non-dystrophic congenital myopathy-is caused by mutations in thin filament genes, of which the nebulin gene is the most frequently affected one. The nebulin gene codes for the giant sarcomeric protein nebulin, which plays a crucial role in skeletal muscle contractile performance. Muscle weakness is a hallmark feature of nemaline myopathy patients with nebulin mutations, and is caused by changes in contractile protein function, including a lower calcium-sensitivity of force generation. To date no therapy exists to treat muscle weakness in nemaline myopathy. Here, we studied the ability of the novel fast skeletal muscle troponin activator, CK-2066260, to augment force generation at submaximal calcium levels in muscle cells from nemaline myopathy patients with nebulin mutations., Methods: Contractile protein function was determined in permeabilised muscle cells isolated from frozen patient biopsies. The effect of 5 μM CK-2066260 on force production was assessed., Results: Nebulin protein concentrations were severely reduced in muscle cells from these patients compared to controls, while myofibrillar ultrastructure was largely preserved. Both maximal active tension and the calcium-sensitivity of force generation were lower in patients compared to controls. Importantly, CK-2066260 greatly increased the calcium-sensitivity of force generation-without affecting the cooperativity of activation-in patients to levels that exceed those observed in untreated control muscle., Conclusions: Fast skeletal troponin activation is a therapeutic mechanism to augment contractile protein function in nemaline myopathy patients with nebulin mutations and with other neuromuscular diseases.

Nebulin--a giant sarcomeric protein--plays a pivotal role in skeletal muscle contractility by specifying thin filament length and function. Although mutations in the gene encoding nebulin (NEB) are a frequent cause of nemaline myopathy, the most common non-dystrophic congenital myopathy, the mechanisms by which mutations in NEB cause muscle weakness remain largely unknown. To better understand these mechanisms, we have generated a mouse model in which Neb exon 55 is deleted (Neb(ΔExon55)) to replicate a founder mutation seen frequently in patients with nemaline myopathy with Ashkenazi Jewish heritage. Neb(ΔExon55) mice are born close to Mendelian ratios, but show growth retardation after birth. Electron microscopy studies show nemaline bodies--a hallmark feature of nemaline myopathy--in muscle fibres from Neb(ΔExon55) mice. Western blotting studies with nebulin-specific antibodies reveal reduced nebulin levels in muscle from Neb(ΔExon55) mice, and immunofluorescence confocal microscopy studies with tropomodulin antibodies and phalloidin reveal that thin filament length is significantly reduced. In line with reduced thin filament length, the maximal force generating capacity of permeabilized muscle fibres and single myofibrils is reduced in Neb(ΔExon55) mice with a more pronounced reduction at longer sarcomere lengths. Finally, in Neb(ΔExon55) mice the regulation of contraction is impaired, as evidenced by marked changes in crossbridge cycling kinetics and by a reduction of the calcium sensitivity of force generation. A novel drug that facilitates calcium binding to the thin filament significantly augmented the calcium sensitivity of submaximal force to levels that exceed those observed in untreated control muscle. In conclusion, we have characterized the first nebulin-based nemaline myopathy model, which recapitulates important features of the phenotype observed in patients harbouring this particular mutation, and which has severe muscle weakness caused by thin filament dysfunction.

Nemaline myopathy is the most common congenital skeletal muscle disease, and mutations in the nebulin gene account for 50% of all cases. Recent studies suggest that the disease severity might be related to the nebulin expression levels. Considering that mutations in the nebulin gene are typically recessive, one would expect that a single functional nebulin allele would maintain nebulin protein expression which would result in preserved skeletal muscle function. We investigated skeletal muscle function of heterozygous nebulin knock-out (i.e., nebulin(+/-)) mice using a multidisciplinary approach including protein and gene expression analysis and combined in vivo and in vitro force measurements. Skeletal muscle anatomy and energy metabolism were studied strictly non-invasively using magnetic resonance imaging and 31P-magnetic resonance spectroscopy. Maximal force production was reduced by around 16% in isolated muscle of nebulin(+/-) mice while in vivo force generating capacity was preserved. Muscle weakness was associated with a shift toward a slower proteomic phenotype, but was not related to nebulin protein deficiency or to an impaired energy metabolism. Further studies would be warranted in order to determine the mechanisms leading to a mild skeletal muscle phenotype resulting from the expression of a single nebulin allele., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The effect of the fast skeletal muscle troponin activator, CK-2066260, on calcium-induced force development was studied in skinned fast skeletal muscle fibers from wildtype (WT) and nebulin deficient (NEB KO) mice. Nebulin is a sarcomeric protein that when absent (NEB KO mouse) or present at low levels (nemaline myopathy (NM) patients with NEB mutations) causes muscle weakness. We studied the effect of fast skeletal troponin activation on WT muscle and tested whether it might be a therapeutic mechanism to increase muscle strength in nebulin deficient muscle. We measured tension-pCa relations with and without added CK-2066260. Maximal active tension in NEB KO tibialis cranialis fibers in the absence of CK-2066260 was ∼60% less than in WT fibers, consistent with earlier work. CK-2066260 shifted the tension-calcium relationship leftwards, with the largest relative increase (up to 8-fold) at low to intermediate calcium levels. This was a general effect that was present in both WT and NEB KO fiber bundles. At pCa levels above ∼6.0 (i.e., calcium concentrations <1 µM), CK-2066260 increased tension of NEB KO fibers to beyond that of WT fibers. Crossbridge cycling kinetics were studied by measuring k(tr) (rate constant of force redevelopment following a rapid shortening/restretch). CK-2066260 greatly increased k(tr) at submaximal activation levels in both WT and NEB KO fiber bundles. We also studied the sarcomere length (SL) dependence of the CK-2066260 effect (SL 2.1 µm and 2.6 µm) and found that in the NEB KO fibers, CK-2066260 had a larger effect on calcium sensitivity at the long SL. We conclude that fast skeletal muscle troponin activation increases force at submaximal activation in both wildtype and NEB KO fiber bundles and, importantly, that this troponin activation is a potential therapeutic mechanism for increasing force in NM and other skeletal muscle diseases with loss of muscle strength.

Niemann-Pick disease type C (NPC) was demonstrated in two successive pregnancies by strongly reduced activity of sphingomyelinase in amniotic fluid cells. By contrast, chorionic villi from the first pregnancy had shown normal sphingomyelinase activity. The prenatal diagnosis of NPC in the two fetuses was confirmed, after termination of the pregnancies, by (phospho)lipid analyses of the fetal livers, by the assay of sphingomyelinase in the fetal fibroblasts and by the demonstration of a defective esterification of exogenous cholesterol and of cholesterol accumulation by filipin staining. Retrospective analysis of cultured amniocytes for cholesterol esterification and filipin staining confirmed the feasibility of these methods for prenatal diagnosis. In a recent pregnancy in the same mother the three available methods were applied to amniotic fluid cells and an unaffected child was correctly predicted. Lipid analysis of liver tissue from the patient with NPC and the two fetuses showed a 3-5 times increased level of cholesterol, a 2-3 times increased level of sphingomyelin and a remarkable increase of bis (monoacylglyceryl) phosphate.

Rat liver mitochondrial phospholipase A2 was purified to near homogeneity by a combination of gel-filtration, hydroxyapatite and Matrex gel Blue A column chromatography. The absolute positional specificity of the enzyme for acylester bonds at the sn-2-position was established in experiments with 1-[9,10-3H2]palmitoyl-2-[1-14C]linoleoylphosphatidylethanolamine. Molecular weight estimations revealed Mr values of 15000 by SDS-polyacrylamide gel electrophoresis and of 9700 gel by gel-filtration over Ultrogel AcA 54 columns. The enzyme is unaffected by diisopropylfluorophosphate and thiol reagents such as 5,5'-dithiobis(2-nitrobenzoic acid), N-ethylmaleimide and iodoacetamide, but is completely inhibited by the alkylating reagent p-bromophenacylbromide.

A comparative study was made of the metal ion requirement of rat liver mitochondrial phospholipase A2 in purified and membrane-associated forms. Membrane-bound enzyme was assayed using either exogenous or endogenous phosphatidylethanolamine. Although several divalent metal ions caused increased activity of the membrane-associated enzyme, only Ca2+ and Sr2+ activated the purified phospholipase A2. The activity in the presence of Sr2+ amounted to about 25% of that found with Ca2+. When the Ca2+ concentration was varied two activity plateaus were observed. The corresponding dissociation constants varied from 6 to 20 microM Ca2+ and from 1.4 to 12 mM Ca2+ for the high- and low-affinity binding sites, respectively, depending on the assay conditions and whether purified or membrane-bound enzyme was used. A kSr2+ of 60 microM was found for the high-affinity binding site. The effect of calmodulin and its antagonist trifluoperazine was also investigated using purified and membrane-associated enzyme. When membrane-bound enzyme was measured with exogenous phosphatidylethanolamine, small stimulations by calmodulin were found. However, these were not believed to indicate a specific role for calmodulin in the Ca2+ dependency of the phospholipase A2, since trifluoperazine did not lower the activity of the membrane-bound enzyme to levels below those found in the presence of Ca2+ alone. Membrane-bound enzyme in its action toward endogenous phosphatidylethanolamine was neither stimulated by calmodulin nor inhibited by trifluoperazine. Purified enzyme was also not stimulated by calmodulin, while trifluoperazine caused small stimulations, presumably due to interactions at the substrate level. These results indicate that calmodulin involvement in phospholipase A2 activation should not be generalized.

Conversion of membrane-bound substrates by membrane-associated enzymes can proceed in principle via intramembrane and intermembrane action. By using rat-liver mitochondria containing labeled phosphatidylethanolamine and inactivated phospholipase A2 as substrate source, and mitochondria containing unlabeled substrate and active enzyme, it is shown that hydrolysis of phosphatidylethanolamine by mitochondrial phospholipase A2 proceeds nearly entirely via intramembrane enzyme action. A study of the characteristics of this mode of enzyme action showed that all mitochondrial phosphoglycerides were hydrolyzed. Plots of approximate initial velocities of hydrolysis against the remaining amounts of each individual phospholipid, indicated that phosphatidylethanolamine was hydrolyzed fastest, with a rate about twice that for phosphatidylcholine and about 10-fold that for cardiolipin. The initial rates remained nearly constant in the initial phase of the hydrolysis, suggesting that the enzyme is surrounded by excess substrate.

The purified A protein and A* protein of bacteriophage phi X174 have been tested for endonuclease activity on single stranded viral phi X174 DNA. The A protein (55.000 daltons) nicks single-stranded DNA in the same way and at the same place as it does superhelical RFI DNA, at the origin of DNA replication. The A* protein (37.000 daltons) can cleave the single-stranded viral DNA at many different sites. It has however a strong preference for the origin of replication. Both proteins generate 3'OH ends and blocked 5' termini at the nick site.