RESUMO
Nebulin, a critical protein of the skeletal muscle thin filament, plays important roles in physiological processes such as regulating thin filament length (TFL), cross-bridge cycling, and myofibril alignment. Pathogenic variants in the nebulin gene (NEB) cause NEB-based nemaline myopathy (NEM2), a genetically heterogeneous disorder characterized by hypotonia and muscle weakness, currently lacking curative therapies. In this study, we examined a cohort of ten NEM2 patients, each with unique pathogenic variants, aiming to understand their impact on mRNA, protein, and functional levels. Results show that pathogenic truncation variants affect NEB mRNA stability and lead to nonsense-mediated decay of the mutated transcript. Moreover, a high incidence of cryptic splice site activation was found in patients with pathogenic splicing variants that are expected to disrupt the actin-binding sites of nebulin. Determination of protein levels revealed patients with either relatively normal or markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in TFL, or reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a pathogenic duplication variant in nebulin that resulted in a four-copy gain in the triplicate region of NEB and a much larger nebulin protein and longer TFL. Additionally, we investigated the effect of Omecamtiv mecarbil (OM), a small-molecule activator of cardiac myosin, on force production of type 1 muscle fibers of NEM2 patients. OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87 to 318%, with the largest effects in patients with the lowest level of nebulin. In summary, this study indicates that post-transcriptional or post-translational mechanisms regulate nebulin expression. Moreover, we propose that the pathomechanism of NEM2 involves not only shortened but also elongated thin filaments, along with the disruption of actin-binding sites resulting from pathogenic splicing variants. Significantly, our findings highlight the potential of OM treatment to improve skeletal muscle function in NEM2 patients, especially those with large reductions in nebulin levels.
Assuntos
Miopatias da Nemalina , Ureia , Humanos , Actinas , Debilidade Muscular , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Miopatias da Nemalina/tratamento farmacológico , Miopatias da Nemalina/genética , Miopatias da Nemalina/patologia , Ureia/análogos & derivados , Proteínas Musculares/genética , Proteínas Musculares/metabolismoRESUMO
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.
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Miopatias da Nemalina , Animais , Camundongos , Fibras Musculares Esqueléticas , Debilidade Muscular/tratamento farmacológico , Debilidade Muscular/genética , Músculo Esquelético/fisiologia , Mutação , Miopatias da Nemalina/tratamento farmacológico , Miopatias da Nemalina/genética , Miostatina/genéticaRESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1007361.].
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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.
Assuntos
Proteínas Musculares , Miopatias da Nemalina , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Animais , Modelos Animais de Doenças , Camundongos , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Miopatias da Nemalina/genética , Miopatias da Nemalina/metabolismo , Sarcômeros/metabolismo , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Knowledge on the genetic epidemiology of disorders in the dog population has implications for both veterinary medicine and sustainable breeding. Limited data on frequencies of genetic disease variants across breeds exists, and the disease heritage of mixed breed dogs remains poorly explored to date. Advances in genetic screening technologies now enable comprehensive investigations of the canine disease heritage, and generate health-related big data that can be turned into action. We pursued population screening of genetic variants implicated in Mendelian disorders in the largest canine study sample examined to date by examining over 83,000 mixed breed and 18,000 purebred dogs representing 330 breeds for 152 known variants using a custom-designed beadchip microarray. We further announce the creation of MyBreedData (www.mybreeddata.com), an online updated inherited disorder prevalence resource with its foundation in the generated data. We identified the most prevalent, and rare, disease susceptibility variants across the general dog population while providing the first extensive snapshot of the mixed breed disease heritage. Approximately two in five dogs carried at least one copy of a tested disease variant. Most disease variants are shared by both mixed breeds and purebreds, while breed- or line-specificity of others is strongly suggested. Mixed breed dogs were more likely to carry a common recessive disease, whereas purebreds were more likely to be genetically affected with one, providing DNA-based evidence for hybrid vigor. We discovered genetic presence of 22 disease variants in at least one additional breed in which they were previously undescribed. Some mutations likely manifest similarly independently of breed background; however, we emphasize the need for follow up investigations in each case and provide a suggested validation protocol for broader consideration. In conclusion, our study provides unique insight into genetic epidemiology of canine disease risk variants, and their relevance for veterinary medicine, breeding programs and animal welfare.
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Doenças do Cão/genética , Cães/genética , Animais , Cruzamento , Bases de Dados Genéticas , Doenças do Cão/epidemiologia , Feminino , Frequência do Gene , Genes Recessivos , Predisposição Genética para Doença , Testes Genéticos/veterinária , Variação Genética , Vigor Híbrido , Masculino , Epidemiologia Molecular , Análise de Sequência com Séries de Oligonucleotídeos/veterinária , Prevalência , Especificidade da EspécieRESUMO
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.
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Actinas/genética , Contração Muscular/fisiologia , Debilidade Muscular/genética , Miopatias Congênitas Estruturais/fisiopatologia , Sarcômeros/patologia , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Debilidade Muscular/fisiopatologia , Músculo Esquelético/patologia , Miopatias Congênitas Estruturais/genética , Sarcômeros/fisiologia , Adulto JovemRESUMO
Nemaline myopathy (NM) is a skeletal muscle disorder caused by mutations in genes that are generally involved in muscle contraction, in particular those related to the structure and/or regulation of the thin filament. Many pathogenic aspects of this disease remain largely unclear. Here, we report novel pathological defects in skeletal muscle fibres of mouse models and patients with NM: irregular spacing and morphology of nuclei; disrupted nuclear envelope; altered chromatin arrangement; and disorganisation of the cortical cytoskeleton. Impairments in contractility are the primary cause of these nuclear defects. We also establish the role of microtubule organisation in determining nuclear morphology, a phenomenon which is likely to contribute to nuclear alterations in this disease. Our results overlap with findings in diseases caused directly by mutations in nuclear envelope or cytoskeletal proteins. Given the important role of nuclear shape and envelope in regulating gene expression, and the cytoskeleton in maintaining muscle fibre integrity, our findings are likely to explain some of the hallmarks of NM, including contractile filament disarray, altered mechanical properties and broad transcriptional alterations.
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Citoesqueleto/patologia , Contração Muscular/fisiologia , Músculo Esquelético/patologia , Miopatias da Nemalina/patologia , Adulto , Idoso , Animais , Núcleo Celular/patologia , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Músculo Esquelético/fisiopatologia , Miopatias da Nemalina/fisiopatologia , Adulto JovemRESUMO
RATIONALE: Diaphragm weakness in critically ill patients prolongs ventilator dependency and duration of hospital stay and increases mortality and healthcare costs. The mechanisms underlying diaphragm weakness include cross-sectional fiber atrophy and contractile protein dysfunction, but whether additional mechanisms are at play is unknown. OBJECTIVES: To test the hypothesis that mechanical ventilation with positive end-expiratory pressure (PEEP) induces longitudinal atrophy by displacing the diaphragm in the caudal direction and reducing the length of fibers. METHODS: We studied structure and function of diaphragm fibers of mechanically ventilated critically ill patients and mechanically ventilated rats with normal and increased titin compliance. MEASUREMENTS AND MAIN RESULTS: PEEP causes a caudal movement of the diaphragm, both in critically ill patients and in rats, and this caudal movement reduces fiber length. Diaphragm fibers of 18-hour mechanically ventilated rats (PEEP of 2.5 cm H2O) adapt to the reduced length by absorbing serially linked sarcomeres, the smallest contractile units in muscle (i.e., longitudinal atrophy). Increasing the compliance of titin molecules reduces longitudinal atrophy. CONCLUSIONS: Mechanical ventilation with PEEP results in longitudinal atrophy of diaphragm fibers, a response that is modulated by the elasticity of the giant sarcomeric protein titin. We postulate that longitudinal atrophy, in concert with the aforementioned cross-sectional atrophy, hampers spontaneous breathing trials in critically ill patients: during these efforts, end-expiratory lung volume is reduced, and the shortened diaphragm fibers are stretched to excessive sarcomere lengths. At these lengths, muscle fibers generate less force, and diaphragm weakness ensues.
Assuntos
Diafragma/patologia , Atrofia Muscular/etiologia , Atrofia Muscular/patologia , Respiração com Pressão Positiva/efeitos adversos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biópsia , Diafragma/diagnóstico por imagem , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Atrofia Muscular/diagnóstico por imagem , Ratos , UltrassonografiaRESUMO
OBJECTIVE: Nemaline myopathy, one of the most common congenital myopathies, is associated with mutations in various genes including ACTA1. This disease is also characterized by various forms/degrees of muscle weakness, with most cases being severe and resulting in death in infancy. Recent findings have provided valuable insight into the underlying pathophysiological mechanisms. Mutations in ACTA1 directly disrupt binding interactions between actin and myosin, and consequently the intrinsic force-generating capacity of muscle fibers. ACTA1 mutations are also associated with variations in myofiber size, the mechanisms of which have been unclear. In the present study, we sought to test the hypotheses that the compromised functional and morphological attributes of skeletal muscles bearing ACTA1 mutations (1) would be directly due to the inefficient actomyosin complex and (2) could be restored by manipulating myosin expression. METHODS: We used a knockin mouse model expressing the ACTA1 His40Tyr actin mutation found in human patients. We then performed in vivo intramuscular injections of recombinant adeno-associated viral vectors harboring a myosin transgene known to facilitate muscle contraction. RESULTS: We observed that in the presence of the transgene, the intrinsic force-generating capacity was restored and myofiber size was normal. INTERPRETATION: This demonstrates a direct link between disrupted attachment of myosin molecules to actin monomers and muscle fiber atrophy. These data also suggest that further therapeutic interventions should primarily target myosin dysfunction to alleviate the pathology of ACTA1-related nemaline myopathy. Ann Neurol 2016;79:717-725.
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Ariel is a mouse mutant that suffers from skeletal muscle myofibrillar degeneration due to the rapid accumulation of large intracellular protein aggregates. This fulminant disease is caused by an ENU-induced recessive mutation resulting in an L342Q change within the motor domain of the skeletal muscle myosin protein MYH4 (MyHC IIb). Although normal at birth, homozygous mice develop hindlimb paralysis from Day 13, consistent with the timing of the switch from developmental to adult myosin isoforms in mice. The mutated myosin (MYH4(L342Q)) is an aggregate-prone protein. Notwithstanding the speed of the process, biochemical analysis of purified aggregates showed the presence of proteins typically found in human myofibrillar myopathies, suggesting that the genesis of ariel aggregates follows a pathogenic pathway shared with other conformational protein diseases of skeletal muscle. In contrast, heterozygous mice are overtly and histologically indistinguishable from control mice. MYH4(L342Q) is present in muscles from heterozygous mice at only 7% of the levels of the wild-type protein, resulting in a small but significant increase in force production in isolated single fibres and indicating that elimination of the mutant protein in heterozygotes prevents the pathological changes observed in homozygotes. Recapitulation of the L342Q change in the functional equivalent of mouse MYH4 in human muscles, MYH1, results in a more aggregate-prone protein.
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Doenças Musculares/genética , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/genética , Sequência de Aminoácidos , Animais , Genes Recessivos , Heterozigoto , Homozigoto , Humanos , Camundongos , Dados de Sequência Molecular , Músculo Esquelético/patologia , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Mutação , Miofibrilas/ultraestrutura , Cadeias Pesadas de Miosina/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Transcrição GênicaRESUMO
Nemaline myopathy, the most common congenital myopathy, is characterized by mutations in genes encoding myofilament proteins such as skeletal α-actin. These mutations are thought to ultimately lead to skeletal muscle weakness. Interestingly, some of the mutations appear to be more potent in males than in females. The underlying mechanisms remain obscure but may be related to sex-specific differences in the myofilament function of both limb and respiratory muscles. To verify this, in the present study, we used skeletal muscles (tibialis anterior and diaphragm) from a transgenic mouse model harbouring the His40Tyr amino acid substitution in skeletal α-actin. In this animal model, 60% of males die by 13weeks of age (the underlying causes of death are obscure but probably due to respiratory insufficiency) whereas females have a normal lifespan. By recording and analysing the mechanics of membrane-permeabilized myofibres, we only observed sex-related differences in the tibialis anterior muscles. Indeed, the concomitant deficits in maximal steady-state isometric force and stiffness of myofibres were less exacerbated in transgenic females than in males, potentially explaining the lower potency in limb muscles. However, the absence of sex-difference in the diaphragm muscles was rather unexpected and suggests that myofilament dysfunction does not solely underlie the sexually dimorphic phenotypes.
Assuntos
Diafragma , Miofibrilas , Miopatias da Nemalina , Caracteres Sexuais , Actinas/genética , Actinas/metabolismo , Animais , Diafragma/metabolismo , Diafragma/patologia , Diafragma/fisiopatologia , Modelos Animais de Doenças , Feminino , Contração Isométrica/genética , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Miofibrilas/genética , Miofibrilas/metabolismo , Miofibrilas/patologia , Miopatias da Nemalina/genética , Miopatias da Nemalina/metabolismo , Miopatias da Nemalina/patologia , Miopatias da Nemalina/fisiopatologia , FenótipoRESUMO
Patients receiving mechanical ventilation in the intensive care unit (ICU) frequently develop contractile weakness of the diaphragm. Consequently, they may experience difficulty weaning from mechanical ventilation, which increases mortality and poses a high economic burden. Because of a lack of knowledge regarding the molecular changes in the diaphragm, no treatment is currently available to improve diaphragm contractility. We compared diaphragm biopsies from ventilated ICU patients (N = 54) to those of non-ICU patients undergoing thoracic surgery (N = 27). By integrating data from myofiber force measurements, x-ray diffraction experiments, and biochemical assays with clinical data, we found that in myofibers isolated from the diaphragm of ventilated ICU patients, myosin is trapped in an energy-sparing, super-relaxed state, which impairs the binding of myosin to actin during diaphragm contraction. Studies on quadriceps biopsies of ICU patients and on the diaphragm of previously healthy mechanically ventilated rats suggested that the super-relaxed myosins are specific to the diaphragm and not a result of critical illness. Exposing slow- and fast-twitch myofibers isolated from the diaphragm biopsies to small-molecule compounds activating troponin restored contractile force in vitro. These findings support the continued development of drugs that target sarcomere proteins to increase the calcium sensitivity of myofibers for the treatment of ICU-acquired diaphragm weakness.
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Diafragma , Contração Muscular , Miosinas , Respiração Artificial , Músculos Respiratórios , Humanos , Animais , Miosinas/metabolismo , Diafragma/metabolismo , Diafragma/fisiopatologia , Músculos Respiratórios/metabolismo , Ratos , Masculino , Unidades de Terapia Intensiva , Pessoa de Meia-Idade , Feminino , Idoso , Hibernação/fisiologia , Actinas/metabolismoRESUMO
Nebulin, a critical protein of the skeletal muscle thin filament, plays important roles in physiological processes such as regulating thin filament length (TFL), cross-bridge cycling, and myofibril alignment. Mutations in the nebulin gene ( NEB ) cause NEB-based nemaline myopathy (NEM2), a genetically heterogeneous disorder characterized by hypotonia and muscle weakness, currently lacking therapies targeting the underlying pathological mechanisms. In this study, we examined a cohort of ten NEM2 patients, each with unique mutations, aiming to understand their impact on mRNA, protein, and functional levels. Results show that truncation mutations affect NEB mRNA stability and lead to nonsense-mediated decay of the mutated transcript. Moreover, a high incidence of cryptic splice site activation was found in patients with splicing mutations which is expected to disrupt the actin-binding sites of nebulin. Determination of protein levels revealed patients with relatively normal nebulin levels and others with markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in TFL, and a positive relation between the reduction in nebulin level and the reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a duplication mutation in nebulin that resulted in a larger nebulin protein and longer TFL. Additionally, we investigated the effect of Omecamtiv mecarbil (OM), a small-molecule activator of cardiac myosin, on force production of type I muscle fibers of NEM2 patients. OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87-318%, with the largest effects in patients with the lowest level of nebulin. In summary, this study indicates that post-transcriptional or post-translational mechanisms regulate nebulin expression. Moreover, we propose that the pathomechanism of NEM2 involves not only shortened but also elongated thin filaments, along with the disruption of actin-binding sites resulting from splicing mutations. Significantly, our findings highlight the potential of OM treatment to improve skeletal muscle function in NEM2 patients, especially those with large reductions in nebulin levels.
RESUMO
Nemaline myopathy (NM) is the most common congenital myopathy and is caused by mutations in various genes including NEB (nebulin), TPM2 (beta-tropomyosin), TPM3 (gamma-tropomyosin), and ACTA1 (skeletal alpha-actin). 20-25% of NM cases carry ACTA1 defects and these particular mutations usually induce substitutions of single residues in the actin protein. Despite increasing clinical and scientific interest, the contractile consequences of these subtle amino acid substitutions remain obscure. To decipher them, in the present study, we originally recorded and analysed the mechanics as well as the X-ray diffraction patterns of human membrane-permeabilized single muscle fibres with a particular peptide substitution in actin, i.e. p.Phe352Ser. Results unravelled an unexpected cascade of molecular and cellular events. During contraction, p.Phe352Ser greatly enhances the strain of individual cross-bridges. Paradoxically, p.Phe352Ser also slightly lowers the number of cross-bridges by altering the rate of myosin head attachment to actin monomers. Overall, at the cell level, these divergent mechanisms conduct to an improved steady-state force production. Such results provide new surprising scientific insights and crucial information for future therapeutic strategies.
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Actinas/genética , Contração Muscular/genética , Mutação/genética , Miopatias da Nemalina/genética , Miopatias da Nemalina/fisiopatologia , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/fisiopatologia , Cadeias Pesadas de Miosina/metabolismo , Estimulação Física , Difração de Raios XRESUMO
BACKGROUND: Whole-body bone scintigraphy is a clinically useful non-invasive and highly sensitive imaging method enabling detection of metabolic changes at an early stage of disease, often earlier than with conventional radiologic procedures. Bone scintigraphy is one of the most common nuclear medicine methods used worldwide. Therefore, it is important that the examination is implemented and performed in an optimal manner giving the patient added value in the subsequent care process. The aim of this national multicentre survey was to investigate Swedish nuclear medicine departments compliance with European practice guidelines for bone scintigraphy. In addition, the effect of image acquisition parameters on the ability to detect metabolic lesions was investigated. METHODS: Twenty-five hospital sites participated in the study. The SIMIND Monte Carlo (MC) simulation and the XCAT phantom were used to simulate ten fictive patient cases with increased metabolic activity distributed at ten different locations in the skeleton. The intensity of the metabolic activity was set into six different levels. Individual simulations were performed for each site, corresponding to their specific camera system and acquisition parameters. Simulated image data sets were then sent to each site and were visually evaluated in terms of if there was one or several locations with increased metabolic activity relative to normal activity. RESULT: There is a high compliance in Sweden with the EANM guidelines regarding image acquisition parameters for whole-body bone scintigraphy. However, up to 40% of the participating sites acquire lower count density in the images than recommended. Despite this, the image quality was adequate to maintain a stable detection level. None of the hospital sites or individual responders deviated according to the statistical analysis. There is a need for at least 2.5 times metabolic activity compared to normal for a lesion to be detected. CONCLUSION: The imaging process is well harmonized throughout the country and there is a high compliance with the EANM guidelines. There is a need for at least 2.5 times the normal metabolic activity for a lesion to be detected as abnormal.
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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 â¼30-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.
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Proteínas Musculares , Peixe-Zebra , Animais , Proteínas Musculares/genética , Músculo Esquelético , SarcômerosRESUMO
Artificial light can be used as a management tool to increase milk yield in dairy production. However, little is known about how cows respond to the spectral composition of light. The aim of this study was to investigate how dairy cows respond to artificial achromatic and chromatic lights. A tie-stall barn equipped with light-emitting diode (LED) light fixtures was used to create the controlled experimental light environments. Two experiments were conducted, both using dairy cows of Swedish Red and light mixtures with red, blue or white light. In experiment I, the response to light of increasing intensity on pupil size was evaluated in five pregnant non-lactating cows. In experiment II 16h of achromatic and chromatic daylight in combination with dim, achromatic night light, was tested on pregnant lactating cows during five weeks to observe long term effects on milk production, activity and circadian rhythms. Particular focus was given to possible carry over effects of blue light during the day on activity at night since this has been demonstrated in humans. Increasing intensity of white and blue light affected pupil size (P<0.001), but there was no effect on pupil size with increased intensity of red light. Milk yield was maintained throughout experiment II, and plasma melatonin was higher during dim night light than in daylight for all treatments (P<0.001). In conclusion, our results show that LED fixtures emitting red light driving the ipRGCs indirectly via ML-cones, blue light stimulating both S-cones and ipRGCs directly and a mixture of wavelengths (white light) exert similar effects on milk yield and activity in tied-up dairy cows. This suggests that the spectral composition of LED lighting in a barn is secondary to duration and intensity.
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Bovinos/sangue , Ritmo Circadiano , Lactação , Luz , Iluminação , Melatonina/biossíntese , Leite/metabolismo , Pupila , Animais , Feminino , Gravidez/sangueRESUMO
Nebulin is a giant protein that winds around the actin filaments in the skeletal muscle sarcomere. Compound-heterozygous mutations in the nebulin gene (NEB) cause typical nemaline myopathy (NM), a muscle disorder characterized by muscle weakness with limited treatment options. We created a mouse model with a missense mutation p.Ser6366Ile and a deletion of NEB exon 55, the Compound-Het model that resembles typical NM. We show that Compound-Het mice are growth-retarded and have muscle weakness. Muscles have a reduced myofibrillar fractional-area and sarcomeres are disorganized, contain rod bodies, and have longer thin filaments. In contrast to nebulin-based severe NM where haplo-insufficiency is the disease driver, Compound-Het mice express normal amounts of nebulin. X-ray diffraction revealed that the actin filament is twisted with a larger radius, that tropomyosin and troponin behavior is altered, and that the myofilament spacing is increased. The unique disease mechanism of nebulin-based typical NM reveals novel therapeutic targets.
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Proteínas Musculares/genética , Mutação de Sentido Incorreto , Miofibrilas/metabolismo , Miopatias da Nemalina/genética , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Animais , Heterozigoto , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Miofibrilas/patologia , Miofibrilas/ultraestrutura , Miopatias da Nemalina/metabolismo , Sarcômeros/metabolismo , Sarcômeros/patologia , Sarcômeros/ultraestrutura , Tropomiosina/química , Tropomiosina/metabolismo , Troponina/química , Troponina/metabolismo , Difração de Raios XRESUMO
Nemaline myopathy (NEM) is a congenital neuromuscular disorder primarily caused by nebulin gene (NEB) mutations. NEM is characterized by muscle weakness for which currently no treatments exist. In NEM patients a predominance of type I fibers has been found. Thus, therapeutic options targeting type I fibers could be highly beneficial for NEM patients. Because type I muscle fibers express the same myosin isoform as cardiac muscle (Myh7), the effect of omecamtiv mecarbil (OM), a small molecule activator of Myh7, was studied in a nebulin-based NEM mouse model (Neb cKO). Skinned single fibers were activated by exogenous calcium and force was measured at a wide range of calcium concentrations. Maximal specific force of type I fibers was much less in fibers from Neb cKO animals and calcium sensitivity of permeabilized single fibers was reduced (pCa50 6.12 ±0.08 (cKO) vs 6.36 ±0.08 (CON)). OM increased the calcium sensitivity of type I single muscle fibers. The greatest effect occurred in type I fibers from Neb cKO muscle where OM restored the calcium sensitivity to that of the control type I fibers. Forces at submaximal activation levels (pCa 6.0-6.5) were significantly increased in Neb cKO fibers (~50%) but remained below that of control fibers. OM also increased isometric force and power during isotonic shortening of intact whole soleus muscle of Neb cKO mice, with the largest effects at physiological stimulation frequencies. We conclude that OM has the potential to improve the quality of life of NEM patients by increasing the force of type I fibers at submaximal activation levels.