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Nemaline myopathy is one of the most common non-dystrophic congenital myopathies. Individuals affected by this condition experience muscle weakness and muscle smallness, often requiring supportive measures like wheelchairs or respiratory support. A significant proportion of patients, approximately one-third, exhibit compound heterozygous nebulin mutations, which usually give rise to the typical form of the disease. Currently, there are no approved treatments available for nemaline myopathy. Our research explored the modulation of myostatin, a negative regulator of muscle mass, in combating the muscle smallness associated with the disease. To investigate the effect of myostatin inhibition, we employed a mouse model with compound heterozygous nebulin mutations that mimic the typical form of the disease. The mice were treated with mRK35, a myostatin antibody, through weekly intraperitoneal injections of 10 mg/kg mRK35, commencing at two weeks of age and continuing until the mice reached four months of age. The treatment resulted in an increase in body weight and an approximate 20% muscle weight gain across most skeletal muscles, without affecting the heart. The minimum Feret diameter of type IIA and IIB fibers exhibited an increase in compound heterozygous mice, while only type IIB fibers demonstrated an increase in wild-type mice. In vitro mechanical experiments conducted on intact extensor digitorum longus muscle revealed that mRK35 augmented the physiological cross-sectional area of muscle fibers and enhanced absolute tetanic force in both wild-type and compound heterozygous mice. Furthermore, mRK35 administration improved grip strength in treated mice. Collectively, these findings indicate that inhibiting myostatin can mitigate the muscle deficits in nebulin-based typical nemaline myopathy, potentially serving as a much-needed therapeutic option. [ABSTRACT FROM AUTHOR]

Abstract Nemaline myopathy (NM) is one of the most common non-dystrophic genetic muscle disorders. NM is often associated with mutations in the NEB gene. Even though the exact NEB-NM pathophysiological mechanisms remain unclear, histological analyses of patients’ muscle biopsies often reveal unexplained accumulation of glycogen and abnormally shaped mitochondria. Hence, the aim of the present study was to define the exact molecular and cellular cascade of events that would lead to potential changes in muscle energetics in NEB-NM. For that, we applied a wide range of biophysical and cell biology assays on skeletal muscle fibres from NM patients as well as untargeted proteomics analyses on isolated myofibres from a muscle-specific nebulin‐deficient mouse model. Unexpectedly, we found that the myosin stabilizing conformational state, known as super-relaxed state, was significantly impaired, inducing an increase in the energy (ATP) consumption of resting muscle fibres from NEB-NM patients when compared with controls or with other forms of genetic/rare, acquired NM. This destabilization of the myosin super-relaxed state had dynamic consequences as we observed a remodeling of the metabolic proteome in muscle fibres from nebulin‐deficient mice. Altogether, our findings explain some of the hitherto obscure hallmarks of NM, including the appearance of abnormal energy proteins and suggest potential beneficial effects of drugs targeting myosin activity/conformations for NEB-NM.

Nemaline myopathy (NM) is one of the most common non-dystrophic genetic muscle disorders. NM is often associated with mutations in the NEB gene. Even though the exact NEB-NM pathophysiological mechanisms remain unclear, histological analyses of patients' muscle biopsies often reveal unexplained accumulation of glycogen and abnormally shaped mitochondria. Hence, the aim of the present study was to define the exact molecular and cellular cascade of events that would lead to potential changes in muscle energetics in NEB-NM. For that, we applied a wide range of biophysical and cell biology assays on skeletal muscle fibres from NM patients as well as untargeted proteomics analyses on isolated myofibres from a muscle-specific nebulin‐deficient mouse model. Unexpectedly, we found that the myosin stabilizing conformational state, known as super-relaxed state, was significantly impaired, inducing an increase in the energy (ATP) consumption of resting muscle fibres from NEB-NM patients when compared with controls or with other forms of genetic/rare, acquired NM. This destabilization of the myosin super-relaxed state had dynamic consequences as we observed a remodeling of the metabolic proteome in muscle fibres from nebulin‐deficient mice. Altogether, our findings explain some of the hitherto obscure hallmarks of NM, including the appearance of abnormal energy proteins and suggest potential beneficial effects of drugs targeting myosin activity/conformations for NEB-NM. [ABSTRACT FROM AUTHOR]

Nemaline myopathy (NM), a structural congenital myopathy, presents a significant clinical and genetic heterogeneity. Here, we compiled molecular and clinical data of 30 Brazilian patients from 25 unrelated families. Next-generation sequencing was able to genetically classify all patients: sixteen families (64%) with mutation in NEB, five (20%) in ACTA1, two (8%) in KLHL40, and one in TPM2 (4%) and TPM3 (4%). In the NEB-related families, 25 different variants, 11 of them novel, were identified; splice site (10/25) and frame shift (9/25) mutations were the most common. Mutation c.24579 G>C was recurrent in three unrelated patients from the same region, suggesting a common ancestor. Clinically, the "typical" form was the more frequent and caused by mutations in the different NM genes. Phenotypic heterogeneity was observed among patients with mutations in the same gene. Respiratory involvement was very common and often out of proportion with limb weakness. Muscle MRI patterns showed variability within the forms and genes, which was related to the severity of the weakness. Considering the high frequency of NEB mutations and the complexity of this gene, NGS tools should be combined with CNV identification, especially in patients with a likely non-identified second mutation. [ABSTRACT FROM AUTHOR]

Nemaline myopathy (NM) is characterized by skeletal muscle weakness and atrophy. No curative treatments exist for this debilitating disease. NM is caused by mutations in proteins involved in thin-filament function, turnover, and maintenance. Mutations in nebulin, encoded by NEB, are the most common cause. Skeletal muscle atrophy is tightly linked to upregulation of MuRF1, an E3 ligase, that targets proteins for proteasome degradation. Here, we report a large increase in MuRF1 protein levels in both patients with nebulin-based NM, also named NEM2, and in mouse models of the disease. We hypothesized that knocking out MuRF1 in animal models of NM with muscle atrophy would ameliorate the muscle deficits. To test this, we crossed MuRF1 KO mice with two NEM2 mouse models, one with the typical form and the other with the severe form. The crosses were viable, and muscles were studied in mice at 3 months of life. Ultrastructural examination of gastrocnemius muscle lacking MuRF1 and with severe NM revealed a small increase in vacuoles, but no significant change in the myofibrillar fractional area. MuRF1 deficiency led to increased weights of various muscle types in the NM models. However, this increase in muscle size was not associated with increased in vivo or in vitro force production. We conclude that knocking out MuRF1 in NEM2 mice increases muscle size, but does not improve muscle function. [ABSTRACT FROM AUTHOR]

Generalized weakness in the pediatric and adolescent population is caused by many disorders that affect the neuromuscular axis. As next-generation sequencing (NGS) is becoming of high yield in replacing more invasive procedures, that is, muscle and nerve biopsy, more previously undiagnosed diseases of the muscles are now labeled with specific pathogenicity. A 16-year-old-girl diagnosed with nemaline myopathy but previously was misdiagnosed with congenital myasthenia and put-on unnecessary medications. Clinicians should be aware of congenital diseases that affect the muscles and know the importance of the NGS in reaching the correct diagnosis more so when there is a history of consanguinity.

Abstract Nemaline myopathy (NM) caused by mutations in the gene encoding nebulin (NEB) accounts for at least 50% of all NM cases worldwide, representing a significant disease burden. Most NEB-NM patients have autosomal recessive disease due to a compound heterozygous genotype. Of the few murine models developed for NEB-NM, most are Neb knockout models rather than harbouring Neb mutations. Additionally, some models have a very severe phenotype that limits their application for evaluating disease progression and potential therapies. No existing murine models possess compound heterozygous Neb mutations that reflect the genotype and resulting phenotype present in most patients. We aimed to develop a murine model that more closely matched the underlying genetics of NEB-NM, which could assist elucidation of the pathogenetic mechanisms underlying the disease. Here, we have characterised a mouse strain with compound heterozygous Neb mutations; one missense (p.Tyr2303His), affecting a conserved actin-binding site and one nonsense mutation (p.Tyr935*), introducing a premature stop codon early in the protein. Our studies reveal that this compound heterozygous model, Neb Y2303H, Y935X, has striking skeletal muscle pathology including nemaline bodies. In vitro whole muscle and single myofibre physiology studies also demonstrate functional perturbations. However, no reduction in lifespan was noted. Therefore, Neb Y2303H,Y935X mice recapitulate human NEB-NM and are a much needed addition to the NEB-NM mouse model collection. The moderate phenotype also makes this an appropriate model for studying NEB-NM pathogenesis, and could potentially be suitable for testing therapeutic applications.

Abstract Background Nebulin is a critical thin filament-binding protein that spans from the Z-disk of the skeletal muscle sarcomere to near the pointed end of the thin filament. Its massive size and actin-binding property allows it to provide the thin filaments with structural and regulatory support. When this protein is lost, nemaline myopathy occurs. Nemaline myopathy causes severe muscle weakness as well as structural defects on a sarcomeric level. There is no known cure for this disease. Methods We studied whether sarcomeric structure and function can be improved by introducing nebulin’s Z-disk region into a nebulin-deficient mouse model (Neb cKO) through adeno-associated viral (AAV) vector therapy. Following this treatment, the structural and functional characteristics of both vehicle-treated and AAV-treated Neb cKO and control muscles were studied. Results Intramuscular injection of this AAV construct resulted in a successful expression of the Z-disk fragment within the target muscles. This expression was significantly higher in Neb cKO mice than control mice. Analysis of protein expression revealed that the nebulin fragment was localized exclusively to the Z-disks and that Neb cKO expressed the nebulin fragment at levels comparable to the level of full-length nebulin in control mice. Additionally, the Z-disk fragment displaced full-length nebulin in control mice, resulting in nemaline rod body formation and a worsening of muscle function. Neb cKO mice experienced a slight functional benefit from the AAV treatment, with a small increase in force and fatigue resistance. Disease progression was also slowed as indicated by improved muscle structure and myosin isoform expression. Conclusions This study reveals that nebulin fragments are well-received by nebulin-deficient mouse muscles and that limited functional benefits are achievable.

Objective: to assess the structural and functional state of skeletal muscles in the hip, as well as changes in the content of the sarcomere cytoskeleton proteins titin and nebulin and their phosphorylation level in patients with chronic alcohol intoxication.Patients and methods. Thirteen patients (4 men and 9 women; mean age, 38.9±9.9 years) with chronic alcohol intoxication were examined. The average duration of regular consumption of alcoholic beverages was 7.6±3.7 years. The mean amount of alcohol consumed per week was 48.2±13.1 units. A control group included 10 healthy volunteers matched for age and sex. A neurological examination was performed according to the generally accepted scheme. Laboratory tests involved blood biochemical analysis estimating the levels of liver enzymes, creatine phosphokinase (CPK), and insulin-like growth factor-1 (IGF-1). All the patients underwent hip magnetic resonance imaging (MRI), followed by assessment of the degree of muscle tissue damage and by determination of the volume of anterior and posterior thigh muscles. The content of titin and nebulin and their phosphorylation level were determined in the muscle tissue samples obtained by an open biopsy from the lateral head of the quadriceps femoris muscle.Results and discussion. Four (30.8%) patients were found to have proximal leg muscle weakness; the degree of paresis was the same in the anterior and posterior thigh muscles. There was a significant increase in the plasma level of liver enzymes; the CPK level remained within the reference values; there was a tendency towards lower IGF-1 levels. Analysis of MRI data showed that 7 (53.8%) patients had fatty degeneration in the thigh muscles. Quantitative evaluation ascertained a significant symmetrical decrease in the volume of anterior thigh muscles and a tendency towards a symmetrical reduction in that in the posterior thigh muscle compared to the control. Analysis of the content of titin and nebulin in the lateral head of the quadriceps femoris muscle revealed a significant decrease in the percentage of nebulin (81.1%; p < 0.01) and intact titin-1 (T1) isoforms (83.6%; p < 0.01). The percentage of proteolytic titin-2 (T2) fragments in the muscle of patients did not differ significantly from that in the control group. Estimating the phosphorylation level of the structural muscle proteins showed no significant differences when compared to the control.Conclusion. Anterior and posterior thigh muscle weakness should be considered as the main clinical manifestation of chronic alcoholic myopathy (CAM) in the absence of biochemical and neurophysiological markers of the disease. Lower extremity muscle MRI that can reveal a lower muscle volume concurrent with fatty degeneration is a non-invasive informative diagnostic technique for CAM. The pathogenesis of skeletal muscle atrophy in chronic alcohol intoxication involves the sarcomere structural proteins titin and nebulin, which regulate the interaction of the major contractile proteins actin and myosin, and whole muscle contraction.

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.

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. [ABSTRACT FROM AUTHOR]

Generalized weakness in the pediatric and adolescent population is caused by many disorders that affect the neuromuscular axis. As next-generation sequencing (NGS) is becoming of high yield in replacing more invasive procedures, that is, muscle and nerve biopsy, more previously undiagnosed diseases of the muscles are now labeled with specific pathogenicity. A 16-year-old-girl diagnosed with nemaline myopathy but previously was misdiagnosed with congenital myasthenia and put-on unnecessary medications. Clinicians should be aware of congenital diseases that affect the muscles and know the importance of the NGS in reaching the correct diagnosis more so when there is a history of consanguinity. [ABSTRACT FROM AUTHOR]

Background: Dilated cardiomyopathy (DCM) is a progressive, lethal disorder that has heterogeneous genetic background. It has been linked to mutations in Nebulin-related-anchoring protein (NRAP) gene. NRAP expressed mainly in striated and cardiac muscles, and it plays substantial role in the sarcomeric contraction cycle and myofibrillogenesis. Case Presentation: A 17-month-old baby girl presented at the age of 13 months with symptoms of heart failure. She was diagnosed as a case of dilated cardiomyopathy. Using whole exome sequencing, diagnosis is confirmed due to homozygous NRAP variant c.400-407 del p.(Cys134 Serfs*12), which create premature stop codon. Conclusion: This case report supports preceding reports that biallelic deletion mutations in NRAP gene cause an autosomal recessive DCM with low penetrance genetic risk factor. However, the age of presentation can vary from early infancy up to adulthood. [JBCGenetics 2018; 1(2.000): 77-80]

Abstract Nemaline myopathies are heterogeneous congenital muscle disorders causing skeletal muscle weakness and, in some cases, death soon after birth. Mutations in nebulin, encoding a large sarcomeric protein required for thin filament function, are responsible for approximately 50% of nemaline myopathy cases. Despite the severity of the disease there is no effective treatment for nemaline myopathy with limited research to develop potential therapies. Several supplements, including L-tyrosine, have been suggested to be beneficial and consequently self-administered by nemaline myopathy patients without any knowledge of their efficacy. We have characterized a zebrafish model for nemaline myopathy caused by a mutation in nebulin. These fish form electron-dense nemaline bodies and display reduced muscle function akin to the phenotypes observed in nemaline myopathy patients. We have utilized our zebrafish model to test and evaluate four treatments currently self-administered by nemaline myopathy patients to determine their ability to increase skeletal muscle function. Analysis of muscle pathology and locomotion following treatment with L-tyrosine, L-carnitine, taurine, or creatine revealed no significant improvement in skeletal muscle function emphasizing the urgency to develop effective therapies for nemaline myopathy.

Nemaline myopathy (NM) caused by mutations in the gene encoding nebulin (NEB) accounts for at least 50% of all NM cases worldwide, representing a significant disease burden. Most NEB-NM patients have autosomal recessive disease due to a compound heterozygous genotype. Of the few murine models developed for NEB-NM, most are Neb knockout models rather than harbouring Neb mutations. Additionally, some models have a very severe phenotype that limits their application for evaluating disease progression and potential therapies. No existing murine models possess compound heterozygous Neb mutations that reflect the genotype and resulting phenotype present in most patients. We aimed to develop a murine model that more closely matched the underlying genetics of NEB-NM, which could assist elucidation of the pathogenetic mechanisms underlying the disease. Here, we have characterised a mouse strain with compound heterozygous Neb mutations; one missense (p.Tyr2303His), affecting a conserved actin-binding site and one nonsense mutation (p.Tyr935*), introducing a premature stop codon early in the protein. Our studies reveal that this compound heterozygous model, NebY2303H, Y935X, has striking skeletal muscle pathology including nemaline bodies. In vitro whole muscle and single myofibre physiology studies also demonstrate functional perturbations. However, no reduction in lifespan was noted. Therefore, NebY2303H,Y935X mice recapitulate human NEB-NM and are a much needed addition to the NEB-NM mouse model collection. The moderate phenotype also makes this an appropriate model for studying NEB-NM pathogenesis, and could potentially be suitable for testing therapeutic applications. [ABSTRACT FROM AUTHOR]

Nemaline myopathy is a rare neuromuscular disorder that affects 1 in 50,000 live births, with prevalence as high as 1 in 20,000 in certain populations. 13 genes have been linked to nemaline myopathy (NM), all of which are associated with the thin filament of the muscle sarcomere. Of the 13 associated genes, mutations in NEBULIN (NEB) accounts for up to 50% of all cases. Currently, the disease is incompletely understood and there are no available therapeutics for patients. To address this urgent need for effective treatments for patients affected by NM, we conducted a large scale chemical screen in a zebrafish model of NEB-related NM and an N-ethyl-N-nitrosourea (ENU)-based genetic screen in a mouse model of NEB exon 55 deletion, the most common NEB mutation in NM patients. Neither screen was able to identify a candidate for therapy development, highlighting the need to transition from conventional chemical therapeutics to gene-based therapies for the treatment of NM.

Purpose: To look for causative genetic mutations in a series of Iranian families with strabismus. In addition, we systematically reviewed all the published articles regarding the role of genetic variations in primary and nonsyndromic comitant strabismus., Methods: Four families with a history of multiple cases of primary and nonsyndromic comitant strabismus were enrolled in this study. Polymerase chain reaction and Sanger sequencing of exons 23, 11, and 3 of the Abelson helper integration site 1 ( AHI1 ), nebulin ( NEB ), and paired box 3 ( PAX3 ) genes were performed, respectively. One offspring of a consanguineous marriage underwent whole-exome sequencing (WES) to look for possible causative variants. To conduct a systematic review, we thoroughly searched PubMed, Scopus, and ISI Web of Knowledge extracting relevant publications, released by April 2021., Results: We examined four Iranian strabismus pedigrees with multiple affected offspring in different generations. Among these 17 participants, 10 family members had strabismus and 7 were healthy. Sanger sequencing did not reveal a causative mutation. Therefore, to further investigate, one affected offspring was chosen for WES. The WES study demonstrated two possible variants in MYO5B and DHODH genes. These genetic variants showed high allele frequency in our population and are thought to be polymorphisms in our series of Iranian families., Conclusions: We demonstrated that mutations in AHI1 , NEB , and PAX3 genes were not common in a series of Iranian patients with familial strabismus. Moreover, by performing WES, we revealed that two variants of uncertain significance as possible causative variants for strabismus are not related to this disease in our population., Competing Interests: There are no conflicts of interest., (Copyright: © 2024 Journal of Current Ophthalmology.)

Background: Nebulin is a critical thin filament-binding protein that spans from the Z-disk of the skeletal muscle sarcomere to near the pointed end of the thin filament. Its massive size and actin-binding property allows it to provide the thin filaments with structural and regulatory support. When this protein is lost, nemaline myopathy occurs. Nemaline myopathy causes severe muscle weakness as well as structural defects on a sarcomeric level. There is no known cure for this disease. Methods: We studied whether sarcomeric structure and function can be improved by introducing nebulin's Z-disk region into a nebulin-deficient mouse model (Neb cKO) through adeno-associated viral (AAV) vector therapy. Following this treatment, the structural and functional characteristics of both vehicle-treated and AAV-treated Neb cKO and control muscles were studied. Results: Intramuscular injection of this AAV construct resulted in a successful expression of the Z-disk fragment within the target muscles. This expression was significantly higher in Neb cKO mice than control mice. Analysis of protein expression revealed that the nebulin fragment was localized exclusively to the Z-disks and that Neb cKO expressed the nebulin fragment at levels comparable to the level of full-length nebulin in control mice. Additionally, the Z-disk fragment displaced full-length nebulin in control mice, resulting in nemaline rod body formation and a worsening of muscle function. Neb cKO mice experienced a slight functional benefit from the AAV treatment, with a small increase in force and fatigue resistance. Disease progression was also slowed as indicated by improved muscle structure and myosin isoform expression. Conclusions: This study reveals that nebulin fragments are well-received by nebulin-deficient mouse muscles and that limited functional benefits are achievable. [ABSTRACT FROM AUTHOR]

Larimichthys crocea nebulin protein was selected to explore novel angiotensin-converting enzyme (ACE) inhibitory peptides. After in silico gastrointestinal proteolysis using pepsin, trypsin and chymotrypsin, several tripeptides were obtained. Then, the cytotoxicity, solubility, bioactivity probability, and adsorption, distribution, metabolism and excretion (ADME) properties of the tripeptides were predicted using online prediction tools. Molecular docking was performed to screen the tightly bound peptides to investigate the interactions between ACE and the novel tripeptides. Finally, potent ACE inhibitory tripeptides were used to explore the active pharmacophores. These results suggested that the tripeptide HGR (His-Gly-Arg) exhibited effective ACE inhibitory activity, with an IC50 value of 106 ± 1.35 µM. The HGRACE complex is stabilised by 14 hydrogen bonds, one attractive charge, and one pi-alkyl interaction. In addition, HGR made contact with the major residues of ACE, i.e., His353, Glu384, Ala354, His513, Tyr523, and Lys511. Furthermore, hydrogen bond acceptors and hydrophobicity are inevitably the most important active features of ACE inhibitory tripeptides, especially hydrogen bond acceptors. [ABSTRACT FROM AUTHOR]

Nebulin (Neb) is associated with the thin filament in skeletal muscle cells, but its functions are not well understood. For this goal, we study skinned slow-twitch soleus muscle fibers from wild-type (Neb+) and conditional Neb knockout (Neb-) mice. We characterize cross-bridge (CB) kinetics and the elementary steps of the CB cycle by sinusoidal analysis during full Ca2+ activation and observe that Neb increases active tension 1.9-fold, active stiffness 2.7-fold, and rigor stiffness 3.0-fold. The ratio of stiffness during activation and rigor states is 62% in Neb+ fibers and 68% in Neb- fibers. These are approximately proportionate to the number of strongly attached CBs during activation. Because the thin filament length is 15% shorter in Neb- fibers than in Neb+ fibers, the increase in force per CB in the presence of Neb is ∼1.5 fold. The equilibrium constant of the CB detachment step (K2), its rate (k2), and the rate of the reverse force generation step (k-4) are larger in Neb+ fibers than in Neb- fibers. The rates of the force generation step (k4) and the reversal detachment step (k-2) change in the opposite direction. These effects can be explained by Le Chatelier's principle: Increased CB strain promotes less force-generating state(s) and/or detached state(s). Further, when CB distributions among the six states are calculated, there is no significant difference in the number of strongly attached CBs between fibers with and without Neb. These results demonstrate that Neb increases force per CB. We also confirm that force is generated by isomerization of actomyosin (AM) from the AM.ADP.Pi state (ADP, adenosine diphophate; Pi, phosphate) to the AM*ADP.Pi state, where the same force is maintained after Pi release to result in the AM*ADP state. We propose that Neb changes the actin (and myosin) conformation for better ionic and hydrophobic/stereospecific AM interaction, and that the effect of Neb is similar to that of tropomyosin. [ABSTRACT FROM AUTHOR]

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 crossbridge 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. [ABSTRACT FROM AUTHOR]

Maintenance of muscle function requires assembly of contractile proteins into highly organized sarcomeres. Mutations in Kelch-like protein 41 (KLHL41) cause nemaline myopathy, a fatal muscle disorder associated with sarcomere disarray. We generated KLHL41 mutant mice, which display lethal disruption of sarcomeres and aberrant expression of muscle structural and contractile proteins, mimicking the hallmarks of the human disease. We show that KLHL41 is poly-ubiquitinated and acts, at least in part, by preventing aggregation and degradation of Nebulin, an essential component of the sarcomere. Furthermore, inhibition of KLHL41 poly-ubiquitination prevents its stabilization of nebulin, suggesting a unique role for ubiquitination in protein stabilization. These findings provide new insights into the molecular etiology of nemaline myopathy and reveal a mechanism whereby KLHL41 stabilizes sarcomeres and maintains muscle function by acting as a molecular chaperone. Similar mechanisms for protein stabilization likely contribute to the actions of other Kelch proteins.

Nemaline myopathy (NM) is a congenital disease that leads to hypotonia and feeding difficulties in neonates. Some cases have a more benign course, with skeletal abnormalities later in life. We analyzed a series of eight patients with NM obtained from a retrospective analysis of 4300 muscle biopsies. Patients were classified as having the typical form in five cases, intermediate form in two cases and severe form in one case. Histochemical analysis showed mixed rods distribution in all cases and predominance of type I fibers in five cases. Immunohistochemical analysis showed abnormal nebulin expression in all patients (four heterogeneous and four absent), homogeneous desmin expression in four cases, strongly positive in three and absent in one, fast myosin expression in a mosaic pattern in six cases and absent in two cases. There was no specific relation between these protein expression patterns and the clinical forms of NM.Miopatia nemalínica (NM) é uma doença congênita que leva a hipotonia e dificuldade de sugar em neonatos. Alguns casos possuem uma evolução benigna, com deformidades ósseas tardias. Nós analisamos uma série de oito pacientes com NM obtidos da análise retrospectiva de 4300 biópsias musculares. Os pacientes foram classificados como forma típica em cinco casos, forma intermediária em dois casos e forma severa em um caso. Análise histoquímica mostrou distribuição mista dos rods em todos os casos e predominância de fibras tipo I em cinco casos. Análise imuno-histoquímica mostrou expressão anormal da nebulina em todos os pacientes (quatro heterogênea e quatro ausente), expressão homogenea da desmina em quatro casos, fortemente positiva em tres e ausente em um, expressão da miosina (rápida) com padrão em mosaico em seis casos e ausente em dois casos. Não há relação específica entre a expressão destas proteínas e as formas clínicas da NM.

Efficient muscle contraction in skeletal muscle is predicated on the regulation of actin filament lengths. In one long-standingmodel thatwas prominent for decades, the giant protein nebulin was proposed to function as a 'molecular ruler' to specify the lengths of the thin filaments. This theory was questioned by many observations, including experiments in which the length of nebulin was manipulated in skeletal myocytes; this approach revealed that nebulin functions to stabilize filamentous actin, allowing thin filaments to reach mature lengths. In addition, more recent data, mostly from in vivo models and identification of new interacting partners, have provided evidence that nebulin is not merely a structural protein. Nebulin plays a role in numerous cellular processes including regulation of muscle contraction, Z-disc formation, andmyofibril organization and assembly. [ABSTRACT FROM AUTHOR]

Several training strategies such as plyometrics have been shown to improve running economy; however, its physiological basis remains elusive. Purpose: To examine the effect of plyometric training on the energy cost of running (ECR, J⋅kg-1⋅min-1), titin, and myosin heavy chain (MHC) isoforms. Methods: Subjects were randomly assigned to a 6-wk plyometric treatment (P; n = 11) or control group (C; n = 11). Preintervention and postintervention outcomes included body composition, vertical jump, sit-and-reach, maximal oxygen consumption (...O2max), speed at onset of blood lactate, 3-km time trial performance, ECR, and a vastus lateralis muscle biopsy for protein analysis. Results: Plyometric intervention resulted in improved time trial (P, 2.6% faster, P = 0.04; C, 1.6%, P = 0.17). ...O2max improved in the P group (5.2%, P = 0.03), whereas the C group increased by 3.1% (P = 0.20). The ECR decreased in the P group as the result of 6 wk of plyometric training (P = 0.02 for stage 3), whereas it increased in the C group (P = 0.02 for stage 3). The ECR correlated strongly with performance at stages 2, 3, and 4 (r > 0.8, P < 0.001) independent of group. There was no significant main effect of group, time, or interaction on any of the protein isoforms analyzed. A negative correlation was found between the ECR at stage 7 and MHC IIa (r = -0.96, P < 0.001), and the ECR at stage 6 with titin isoform 1 (T1)/T2 ratio (r = -0.69, P = 0.007) independent of group. Conclusion: Six weeks of plyometric training improved running performance and the ECR despite no measurable changes in MHC and titin isoforms. However, higher MHC IIa and lower T1/T2 isoform ratios correlated to lower ECR. [ABSTRACT FROM AUTHOR]

Background Congenital myopathies (CM) are a group of rare inherited muscle disorders characterized by particular histopathological alterations on muscle biopsy. Core-rod myopathy is a CM presenting with cores and rods as distinctive muscle morphological features. Methods/results: We describe 3 young patients presenting congenital core-rod myopathy with bilateral foot-drop associated with autosomal recessive nebulin gene ( NEB ) mutations detected by exome sequencing. Conclusions This report illustrates that core-rod congenital myopathy with foot-drop is frequently associated with NEB gene mutations and should be considered in the differential diagnosis of early onset distal myopathies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Several dog breeds are susceptible to developing primary angle closure glaucoma (PACG), which suggests a genetic basis for the disease. We have identified a four-generation Basset Hound pedigree with characteristic autosomal recessive PACG that closely recapitulates PACG in humans. Our aim is to utilize gene mapping and whole exome sequencing approaches to identify PACG-causing sequence variants in the Basset. Extensive clinical phenotyping of all pedigree members was conducted. SNP-chip genotyping was carried out in 9 affected and 15 unaffected pedigree members. Two-point and multipoint linkage analyses of genome-wide SNP data were performed using Superlink-Online SNP-1.1 and a locus was mapped to chromosome 19q with a maximum LOD score of 3.24. The locus contains 12 Ensemble predicted canine genes and is syntenic to a region on chromosome 2 in the human genome. Using exome-sequencing analysis, a possibly damaging, non-synonymous variant in the gene Nebulin (NEB) was found to segregate with PACG which alters a phylogenetically conserved Lysine residue. The association of this variants with PACG was confirmed in a secondary cohort of unrelated Basset Hounds (p = 3.4 × 10-4, OR = 15.3 for homozygosity). Nebulin, a protein that promotes the contractile function of sarcomeres, was found to be prominently expressed in the ciliary muscles of the anterior segment. Our findings may provide insight into the molecular mechanisms that underlie PACG. The phenotypic similarities of disease presentation in dogs and humans may enable the translation of findings made in this study to patients with PACG. [ABSTRACT FROM AUTHOR]

Giant muscle proteins (e.g. titin, nebulin, and obscurin) play a seminal role in muscle elasticity, stretch response, and sarcomeric organization. Each giant protein consists of multiple tandem structural domains, usually arranged in a modular fashion ranging from 500 kDa to 4 MDa. Although many of the domains are similar in structure, subtle differences create a unique function of each domain. Recent high and low resolution structural and dynamic studies now suggest more nuanced overall protein structures than previously realized. These findings show that atomic structure, interactions between tandem domains, and intrasarcomeric environment all influence the shape, motion, and therefore function of giant proteins. In this article we will review the current understanding of titin, obscurin, and nebulin structure, from the atomic level through the molecular level.

We examined a Korean family with complex phenotypes characterized by intellectual disability, epilepsy and early-childhood-onset generalized muscle weakness. Since we did not find any abnormality using several conventional genetic tests for detection of chromosomal aberrations, gene copy number variations and mitochondrial gene mutations, we aimed to identify disease-causing genetic alteration(s) in this family. We conducted whole-exome sequencing (WES) in this family. After filtering the WES data, we compared five exome sequences of two affected siblings, one unaffected sibling and the unaffected parents, and we determined the allele frequency of the identified variants in an Asian population. Finally, we selected one candidate variant pair which is unique in the patients and corresponds to an autosomal recessive genetic model. The two affected siblings had the same compound heterozygous variation in the NEB gene encoding nebulin, which was composed of two different missense variants: c.2603T>C (p.L868P) in exon 27 and c.21340C>T (p.R7114W) in exon 143. We confirmed these variations by Sanger sequencing. On the basis of the fundamental role of nebulin in the brain and skeletal muscles, we concluded that this compound heterozygous NEB variation may be a sound candidate for the disease-causing mutation in this family. Since the patients are characterized by generalized muscle weakness together with neurodevelopmental phenotypes, it is suggested that NEB mutations could manifest more diverse phenotypes than those previously described. [ABSTRACT FROM AUTHOR]

Mutations in nebulin, a giant muscle protein with 185 actin-binding nebulin repeats, are the major cause of nemaline myopathy in humans. Nebulin sets actin thin filament length in sarcomeres, potentially by stabilizing thin filaments in the I-band, where nebulin and thin filaments coalign. However, the precise role of nebulin in setting thin filament length and its other functions in regulating power output are unknown. Here, we show that Lasp, the only member of the nebulin family in Drosophila melanogaster, acts at two distinct sites in the sarcomere and controls thin filament length with just two nebulin repeats. We found that Lasp localizes to the Z-disc edges to control I-band architecture and also localizes at the A-band, where it interacts with both actin and myosin to set proper filament spacing. Furthermore, introducing a single amino acid change into the two nebulin repeats of Lasp demonstrated different roles for each domain and established Lasp as a suitable system for studying nebulin repeat function. [ABSTRACT FROM AUTHOR]

Nemaline myopathy (NM) is a congenital myopathy that can result in lethal muscle dysfunction and is thought to be a disease of the sarcomere thin filament. Recently, several proteins of unknown function have been implicated in NM, but the mechanistic basis of their contribution to disease remains unresolved. Here, we demonstrated that loss of a muscle-specific protein, kelch-like family member 40 (KLHL40), results in a nemaline-like myopathy in mice that closely phenocopies muscle abnormalities observed in KLHL40-deficient patients. We determined that KLHL40 localizes to the sarcomere I band and A band and binds to nebulin (NEB), a protein frequently implicated in NM, as well as a putative thin filament protein, leiomodin 3 (LMOD3). KLHL40 belongs to the BTB-BACK-kelch (BBK) family of proteins, some of which have been shown to promote degradation of their substrates. In contrast, we found that KLHL40 promotes stability of NEB and LMOD3 and blocks LMOD3 ubiquitination. Accordingly, NEB and LMOD3 were reduced in skeletal muscle of both Klhl40-/- mice and KLHL40-deficient patients. Loss of sarcomere thin filament proteins is a frequent cause of NM; therefore, our data that KLHL40 stabilizes NEB and LMOD3 provide a potential basis for the development of NM in KLHL40-deficient patients. [ABSTRACT FROM AUTHOR]