RESUMO
Crotamine and crotamine-like peptides are non-enzymatic polypeptides, belonging to the family of myotoxins, which are found in high concentration in the venom of the Crotalus genus. Helleramine was isolated and purified from the venom of the Southern Pacific rattlesnake, Crotalus oreganus helleri. This peptide had a similar, but unique, identity to crotamine and crotamine-like proteins isolated from other rattlesnakes species. The variability of crotamine-like protein amino acid sequences may allow different toxic effects on biological targets or optimize the action against the same target of different prey. Helleramine was capable of increasing intracellular Ca2+ in Chinese Hamster Ovary (CHO) cell line. It inhibited cell migration as well as cell viability (IC50 = 11.44 µM) of C2C12, immortalized skeletal myoblasts, in a concentration dependent manner, and promoted early apoptosis and cell death under our experimental conditions. Skeletal muscle harvested from mice 24 h after helleramine injection showed contracted myofibrils and profound vacuolization that enlarged the subsarcolemmal space, along with loss of plasmatic and basal membrane integrity. The effects of helleramine provide further insights and evidence of myotoxic activities of crotamine-like peptides and their possible role in crotalid envenomings.
Assuntos
Venenos de Crotalídeos/farmacologia , Crotalus , Placa Motora/efeitos dos fármacos , Músculo Estriado/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Células CHO , Linhagem Celular , Cricetulus , Camundongos , Placa Motora/ultraestrutura , Músculo Estriado/ultraestrutura , PeptídeosRESUMO
Troponin is an essential component of striated muscle and it regulates the sliding of actomyosin system in a calcium-dependent manner. Despite its importance, the structure of troponin has been elusive due to its high structural heterogeneity. In this study, we analyzed the 3D structures of murine cardiac thin filaments using a cryo-electron microscope equipped with a Volta phase plate (VPP). Contrast enhancement by a VPP enabled us to reconstruct the entire repeat of the thin filament. We determined the orientation of troponin relative to F-actin and tropomyosin, and characterized the interactions between troponin and tropomyosin. This study provides a structural basis for understanding the molecular mechanism of actomyosin system.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Actinas/ultraestrutura , Músculo Estriado/ultraestrutura , Troponina/ultraestrutura , Actinas/química , Actomiosina/química , Actomiosina/ultraestrutura , Animais , Cálcio , Microscopia Crioeletrônica , Camundongos , Sarcômeros/química , Sarcômeros/ultraestrutura , Tropomiosina/ultraestrutura , Troponina/químicaRESUMO
Muscular contraction is a fundamental phenomenon in all animals; without it life as we know it would be impossible. The basic mechanism in muscle, including heart muscle, involves the interaction of the protein filaments myosin and actin. Motility in all cells is also partly based on similar interactions of actin filaments with non-muscle myosins. Early studies of muscle contraction have informed later studies of these cellular actin-myosin systems. In muscles, projections on the myosin filaments, the so-called myosin heads or cross-bridges, interact with the nearby actin filaments and, in a mechanism powered by ATP-hydrolysis, they move the actin filaments past them in a kind of cyclic rowing action to produce the macroscopic muscular movements of which we are all aware. In this special issue the papers and reviews address different aspects of the actin-myosin interaction in muscle as studied by a plethora of complementary techniques. The present overview provides a brief and elementary introduction to muscle structure and function and the techniques used to study it. It goes on to give more detailed descriptions of what is known about muscle components and the cross-bridge cycle using structural biology techniques, particularly protein crystallography, electron microscopy and X-ray diffraction. It then has a quick look at muscle mechanics and it summarises what can be learnt about how muscle works based on the other studies covered in the different papers in the special issue. A picture emerges of the main molecular steps involved in the force-producing process; steps that are also likely to be seen in non-muscle myosin interactions with cellular actin filaments. Finally, the remarkable advances made in studying the effects of mutations in the contractile assembly in causing specific muscle diseases, particularly those in heart muscle, are outlined and discussed.
Assuntos
Actinas/metabolismo , Músculos/fisiologia , Miosinas/metabolismo , Actinas/química , Animais , Humanos , Modelos Biológicos , Contração Muscular , Músculo Estriado/fisiologia , Músculo Estriado/ultraestrutura , Músculos/ultraestrutura , Miosinas/química , Ligação Proteica , Sarcômeros/metabolismo , Relação Estrutura-AtividadeRESUMO
The ultrastructure of mitochondria in the flattened circomyarian fibers of the horsehair worm Gordionus alpestris (Nemathelminthes) was examined. In contrast to the previously published data, we showed these mitochondria to be giant elongated organelles that densely fill the central cytoplasmic space of the ribbon-like muscle fibers. No fundamental differences were found in the ultrastructure of the muscle tissue mitochondria in actively moving free-living and parasitic G. alpestris worms. The functional significance of the observed ultrastructural organization of mitochondria is discussed in connection with the necessity for an extended mitochondrial membrane system for a uniform supply of active muscle tissue with energy.
Assuntos
Helmintos/anatomia & histologia , Mitocôndrias/ultraestrutura , Músculo Estriado/ultraestrutura , Animais , Metabolismo Energético , Helmintos/citologia , Mitocôndrias Musculares , Membranas MitocondriaisRESUMO
Titin, the largest protein known, forms an elastic myofilament in the striated muscle sarcomere. To establish titin's contribution to skeletal muscle passive stiffness, relative to that of the extracellular matrix, a mouse model was created in which titin's molecular spring region was shortened by deleting 47 exons, the TtnΔ112-158 model. RNA sequencing and super-resolution microscopy predicts a much stiffer titin molecule. Mechanical studies with this novel mouse model support that titin is the main determinant of skeletal muscle passive stiffness. Unexpectedly, the in vivo sarcomere length working range was shifted to shorter lengths in TtnΔ112-158 mice, due to a ~ 30% increase in the number of sarcomeres in series (longitudinal hypertrophy). The expected effect of this shift on active force generation was minimized through a shortening of thin filaments that was discovered in TtnΔ112-158 mice. Thus, skeletal muscle titin is the dominant determinant of physiological passive stiffness and drives longitudinal hypertrophy. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).
Assuntos
Conectina/química , Hipertrofia/genética , Músculo Esquelético/ultraestrutura , Músculo Estriado/ultraestrutura , Citoesqueleto de Actina/química , Citoesqueleto de Actina/genética , Animais , Conectina/genética , Tecido Elástico/química , Matriz Extracelular/química , Matriz Extracelular/genética , Humanos , Hipertrofia/fisiopatologia , Camundongos , Músculo Esquelético/química , Músculo Estriado/química , Músculo Estriado/fisiologia , Miocárdio/química , Miocárdio/patologia , Miofibrilas/química , Sarcômeros/química , Sarcômeros/genéticaRESUMO
One of the most iconic images in biology is the cross-striated appearance of a skeletal muscle fiber. The repeating band pattern shows that all of the sarcomeres are the same length. All of the A bands are the same length and are located in the middle of the sarcomeres. Furthermore, all of the myofibrils are transversely aligned across the muscle fiber. It has been known for 300 yr that skeletal muscle is striated, but only in the last 40 yr has a molecular understanding of the striations emerged. In the 1950s it was discovered that the extraction of myosin from myofibrils abolished the A bands, and the myofibrils were no longer striated. With the further extraction of actin, only the Z disks remained. Strangely, the sarcomere length did not change, and these "ghost" myofibrils still exhibited elastic behavior. The breakthrough came in the 1970s with the discovery of the gigantic protein titin. Titin, an elastic protein ~1 µm in length, runs from the Z disk to the middle of the A band and ensures that each sarcomere is the same length. Titin anchors the A band in the middle of the sarcomere and may determine thick-filament length and thus A-band length. In the 1970s it was proposed that the intermediate filament desmin, which surrounds the Z disks, connects adjacent myofibrils, resulting in the striated appearance of a skeletal muscle fiber.
Assuntos
Pesquisa Biomédica/história , Citoesqueleto/fisiologia , Músculo Esquelético/fisiologia , Sarcômeros/fisiologia , Animais , Citoesqueleto/ultraestrutura , História do Século XVII , História do Século XVIII , História do Século XIX , História do Século XX , História do Século XXI , Humanos , Músculo Esquelético/ultraestrutura , Músculo Estriado/fisiologia , Músculo Estriado/ultraestrutura , Sarcômeros/ultraestruturaRESUMO
BACKGROUND: Peripheral artery disease (PAD) is a vascular disease caused by atherosclerosis, resulting in decreased blood flow to the lower extremities. The ankle-brachial index (ABI) is a standard PAD diagnostic test but only identifies reduced blood flow based on blood pressure differences. The early signs of PAD manifest themselves not only at a clinical level but also at an elemental and biochemical level. However, the biochemical and elemental alterations to PAD muscle are not well understood. The objective of this study was to compare fundamental changes in intracellular elemental compositions between control, claudicating, and critical limb ischemia muscle tissue. MATERIALS AND METHODS: Gastrocnemius biopsies from three subjects including one control (ABI ≥ 0.9), one claudicating (0.4 ≤ ABI < 0.9), and one critical limb ischemia patient (ABI < 0.4) were evaluated using a scanning electron microscope and energy dispersive X-ray spectroscopy to quantify differences in elemental compositions. Spectra were collected for five myofibers per specimen. An analysis of variance was performed to identify significant differences in muscle elemental compositions. RESULTS: This study revealed that intracellular magnesium and calcium were lower in PAD compared with control myofibers, whereas sulfur was higher. Magnesium and calcium are antagonistic, meaning, if magnesium concentrations go down calcium concentrations should go up. However, our findings do not support this antagonism in PAD. Our analysis found decreases in sodium and potassium, in PAD myofibers. CONCLUSIONS: These findings may provide insight into the pathologic mechanisms that may operate in ischemic muscle and aid in the development of specialized preventive and rehabilitative treatment plans for PAD patients.
Assuntos
Claudicação Intermitente/diagnóstico , Isquemia/diagnóstico , Músculo Estriado/irrigação sanguínea , Doença Arterial Periférica/diagnóstico , Idoso , Índice Tornozelo-Braço , Biópsia , Progressão da Doença , Eletrólitos/análise , Humanos , Extremidade Inferior , Masculino , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Músculo Estriado/metabolismo , Músculo Estriado/patologia , Músculo Estriado/ultraestrutura , Doença Arterial Periférica/complicações , Doença Arterial Periférica/patologia , Fatores de Risco , Espectrometria por Raios XRESUMO
The purpose of the present work was to study the effect of low-level laser therapy (LLLT): helium-neon (He-Ne) and gallium arsenide (Ga-As) laser on the histomorphology of muscle and mitochondria in experimental myopathy in rats. Thirty Suquía strain female rats were distributed in groups: (A) control (intact), (B) injured, (C) injured and treated with He-Ne laser, (D) injured and treated with Ga-As laser, (E) irradiated with He-Ne laser on the non-injured muscle, and (F) irradiated with Ga-As laser on the non-injured muscle. Myopathy was induced by injecting 0.05 mg/rat/day of adrenaline in the left gastrocnemius muscle at the same point on five consecutive days, in groups B, C, and D. LLLT was applied with 9.5 J cm-2 daily for seven consecutive days in groups C, D, E, and F. The muscles were examined with optic and electronic microscopy. The inflammation was classified as absent, mild, and intense and the degree of mitochondrial alteration was graded I, II, III, and IV. Categorical data were statistically analyzed by Chi-square and the Fisher-Irwin Bilateral test, setting significant difference at p < 0.05. The damage found in muscle and mitochondria histomorphology in animals with induced myopathy (B) was intense or severe inflammation with grade III or IV of mitochondrial alteration. They underwent significant regression (p < 0.001) compared with the groups treated with He-Ne (C) and Ga-As (D) laser, in which mild or moderate inflammation was seen and mitochondrial alteration grades I and II, recovering normal myofibrillar architecture. No differences were found between the effects caused by the two lasers, or between groups A, E, and F. Group A was found to be different from B, C, and D (p < 0.001). LLLT in experimental myopathy caused significant muscular and mitochondrial morphologic recovery.
Assuntos
Terapia com Luz de Baixa Intensidade , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Doenças Musculares/patologia , Doenças Musculares/radioterapia , Animais , Feminino , Lasers de Gás , Lasers Semicondutores , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Músculo Estriado/patologia , Músculo Estriado/ultraestrutura , RatosRESUMO
In recent years, rapid discoveries have been made relating to Ca2+ handling at specific organelles that have important implications for whole-cell Ca2+ homeostasis. In particular, the structures of the endoplasmic reticulum (ER) Ca2+ channels revealed by electron cryomicroscopy (cryo-EM), continuous updates on the structure, regulation, and role of the mitochondrial calcium uniporter (MCU) complex, and the analysis of lysosomal Ca2+ signaling are milestones on the route towards a deeper comprehension of the complexity of global Ca2+ signaling. In this review we summarize recent discoveries on the regulation of interorganellar Ca2+ homeostasis and its role in pathophysiology.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica/fisiologia , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Homeostase , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Músculo Estriado/metabolismo , Músculo Estriado/ultraestrutura , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismoRESUMO
As the only striated muscle tissues in the body, skeletal and cardiac muscle share numerous structural and functional characteristics, while exhibiting vastly different size and regenerative potential. Healthy skeletal muscle harbors a robust regenerative response that becomes inadequate after large muscle loss or in degenerative pathologies and aging. In contrast, the mammalian heart loses its regenerative capacity shortly after birth, leaving it susceptible to permanent damage by acute injury or chronic disease. In this review, we compare and contrast the physiology and regenerative potential of native skeletal and cardiac muscles, mechanisms underlying striated muscle dysfunction, and bioengineering strategies to treat muscle disorders. We focus on different sources for cellular therapy, biomaterials to augment the endogenous regenerative response, and progress in engineering and application of mature striated muscle tissues in vitro and in vivo. Finally, we discuss the challenges and perspectives in translating muscle bioengineering strategies to clinical practice.
Assuntos
Músculo Estriado/patologia , Músculo Estriado/fisiopatologia , Regeneração , Cicatrização , Animais , Humanos , Modelos Biológicos , Músculo Estriado/ultraestrutura , Doenças Musculares/fisiopatologia , Doenças Musculares/terapia , Engenharia TecidualRESUMO
Cyclophilin D (CyP-D) is the mitochondrial-specific member of the evolutionally conserved cyclophilin family, and plays an important role in the regulation of mitochondrial permeability transition (MPT) under stress. Recently we have demonstrated that respiratory mitochondria undergo mitochondrial flash ("mitoflash") activity which is coupled with transient MPT under physiological conditions. However, whether and how CyP-D regulates mitoflashes remain incompletely understood. By using both loss- and gain-of-function approaches in isolated cardiomyocytes, beating hearts, and skeletal muscles in living mice, we revisited the role of CyP-D in the regulation of mitoflashes. Overexpression of CyP-D increased, and knockout of it halved, cardiac mitoflash frequency, while mitoflash amplitude and kinetics remained unaffected. However, CyP-D ablation did not alter mitoflash frequency, with mitoflash amplitude increased, in gastrocnemius muscles. This disparity was accompanied by 4-fold higher CyP-D expression in mouse cardiac than skeletal muscles. The mitochondrial maximal respiration rate and reserved capacity were reduced in CyP-D-null cardiomyocytes. These data indicate that CyP-D is a significant regulator of mitoflash ignition and mitochondrial metabolism in heart. In addition, tissue-specific CyP-D expression may partly explain the differential regulation of mitoflashes in the two types of striated muscles.
Assuntos
Ciclofilinas/metabolismo , Mitocôndrias/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Peptidil-Prolil Isomerase F , Ciclofilinas/genética , Feminino , Regulação da Expressão Gênica , Genes Reporter , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Músculo Estriado/metabolismo , Músculo Estriado/ultraestrutura , Miocárdio/ultraestrutura , Miócitos Cardíacos/ultraestrutura , Técnicas de Cultura de Órgãos , Especificidade de Órgãos , Cultura Primária de Células , Transdução de SinaisRESUMO
According to the current opinion, lymph-heart striated muscle represents a specialized type of skeletal muscles in frogs. Here, we studied muscle fibers in mechanically damaged lymph hearts during the first postoperative week using electron-microscopic autoradiography. We present evidence that both, the satellite cells and pre-existing muscle fibers bordering the site of injury, contribute directly to the lymph-heart muscle regeneration. Several muscle fibers located in the vicinity of the damaged area displayed features of nuclear and sarcoplasmic activation. We also observed ultrastructural changes indicating activation of a few satellite cells, namely decondensation of chromatin, enlargement of nuclei and nucleoli, appearance of free ribosomes and rough endoplasmic reticulum tubules in the cytoplasm. Electron-microscopic autoradiography showed that 4 h after single (3)H-thymidine administration on the seventh day after injury not only the activated satellite cells, but also some nuclei of myofibers bordering the injured zone are labeled. We showed that both, the myonuclei of fibers displaying the signs of degenerative/reparative processes in the sarcoplasm and the myonuclei of the fibers enriched with highly organized myofibrils, can re-enter into the S-phase. Our results indicate that the nuclei of lymph-heart myofibers can reactivate DNA synthesis during regenerative myogenesis, unlike the situation in regenerating frog skeletal muscle where myogenic cells do not synthesize DNA at the onset of myofibrillogenesis.
Assuntos
Músculo Estriado/ultraestrutura , Animais , Núcleo Celular/ultraestrutura , Vasos Linfáticos/citologia , Desenvolvimento Muscular , Músculo Estriado/diagnóstico por imagem , Músculo Estriado/fisiologia , Radiografia , Rana temporaria , RegeneraçãoRESUMO
Muscle contraction results from cyclic interactions between the contractile proteins myosin and actin, driven by the turnover of adenosine triphosphate (ATP). Despite intense studies, several molecular events in the contraction process are poorly understood, including the relationship between force-generation and phosphate-release in the ATP-turnover. Different aspects of the force-generating transition are reflected in the changes in tension development by muscle cells, myofibrils and single molecules upon changes in temperature, altered phosphate concentration, or length perturbations. It has been notoriously difficult to explain all these events within a given theoretical framework and to unequivocally correlate observed events with the atomic structures of the myosin motor. Other incompletely understood issues include the role of the two heads of myosin II and structural changes in the actin filaments as well as the importance of the three-dimensional order. We here review these issues in relation to controversies regarding basic physiological properties of striated muscle. We also briefly consider actomyosin mutation effects in cardiac and skeletal muscle function and the possibility to treat these defects by drugs.
Assuntos
Trifosfato de Adenosina/metabolismo , Contração Muscular , Músculo Estriado/metabolismo , Miosinas/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Humanos , Músculo Estriado/fisiologia , Músculo Estriado/ultraestrutura , Miosinas/ultraestruturaRESUMO
Muscle nuclei are exposed to variable cytoplasmic strain produced by muscle contraction and relaxation, but their morphology remains stable. Still, the mechanism responsible for maintaining myonuclear architecture, and its importance, is currently elusive. Herein, we uncovered a unique myonuclear scaffold in Drosophila melanogaster larval muscles, exhibiting both elastic features contributed by the stretching capacity of MSP300 (nesprin) and rigidity provided by a perinuclear network of microtubules stabilized by Shot (spectraplakin) and EB1. Together, they form a flexible perinuclear shield that protects myonuclei from intrinsic or extrinsic forces. The loss of this scaffold resulted in significantly aberrant nuclear morphology and subsequently reduced levels of essential nuclear factors such as lamin A/C, lamin B, and HP1. Overall, we propose a novel mechanism for maintaining myonuclear morphology and reveal its critical link to correct levels of nuclear factors in differentiated muscle fibers. These findings may shed light on the underlying mechanism of various muscular dystrophies.
Assuntos
Núcleo Celular/fisiologia , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Musculares/fisiologia , Actinas/metabolismo , Animais , Drosophila melanogaster/ultraestrutura , Elasticidade , Laminas/metabolismo , Larva/metabolismo , Larva/ultraestrutura , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/ultraestrutura , Músculo Estriado/metabolismo , Músculo Estriado/ultraestrutura , Transporte ProteicoRESUMO
Changes in isoform composition, gene expression of titin and nebulin, and isoform composition of myosin heavy chains as well as changes in titin phosphorylation level in skeletal (m. gastrocnemius, m. tibialis anterior, and m. psoas) and cardiac muscles of mice were studied after a 30-day-long space flight onboard the Russian spacecraft "BION-M" number 1. A muscle fibre-type shift from slow-to-fast and a decrease in the content of titin and nebulin in the skeletal muscles of animals from "Flight" group was found. Using Pro-Q Diamond staining, an ~3-fold increase in the phosphorylation level of titin in m. gastrocnemius of mice from the "Flight" group was detected. The content of titin and its phosphorylation level in the cardiac muscle of mice from "Flight" and "Control" groups did not differ; nevertheless an increase (2.2 times) in titin gene expression in the myocardium of flight animals was found. The observed changes are discussed in the context of their role in the contractile activity of striated muscles of mice under conditions of weightlessness.
Assuntos
Citoesqueleto de Actina/genética , Regulação da Expressão Gênica , Músculo Estriado/metabolismo , Cadeias Pesadas de Miosina/genética , Voo Espacial , Citoesqueleto de Actina/metabolismo , Animais , Conectina/genética , Conectina/metabolismo , Densitometria , Eletroforese em Gel de Poliacrilamida , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Estriado/ultraestrutura , Cadeias Pesadas de Miosina/metabolismo , Tamanho do Órgão , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sarcômeros/metabolismo , Sarcômeros/ultraestruturaRESUMO
The transverse tubule system in mammalian striated muscle is highly organized and contributes to optimal and homogeneous contraction. Diverse pathologies such as heart failure and atrial fibrillation include disorganization of t-tubules and contractile dysfunction. Few tools are available for the quantification of the organization of the t-tubule system. We developed a plugin for the ImageJ/Fiji image analysis platform developed by the National Institutes of Health. This plugin (TTorg) analyzes raw confocal microscopy images. Analysis options include the whole image, specific regions of the image (cropping), and z-axis analysis of the same image. Batch analysis of a series of images with identical criteria is also one of the options. There is no need to either reorientate any specimen to the horizontal or to do a thresholding of the image to perform analysis. TTorg includes a synthetic "myocyte-like" image generator to test the plugin's efficiency in the user's own experimental conditions. This plugin was validated on synthetic images for different simulated cell characteristics and acquisition parameters. TTorg was able to detect significant differences between the organization of the t-tubule systems in experimental data of mouse ventricular myocytes isolated from wild-type and dystrophin-deficient mice. TTorg is freely distributed, and its source code is available. It provides a reliable, easy-to-use, automatic, and unbiased measurement of t-tubule organization in a wide variety of experimental conditions.
Assuntos
Algoritmos , Análise de Fourier , Miócitos Cardíacos/ultraestrutura , Software , Animais , Processamento de Imagem Assistida por Computador/métodos , Camundongos , Camundongos Knockout , Microscopia Confocal/métodos , Músculo Estriado/ultraestruturaRESUMO
Myosin interacting-heads (MIH) motifs are visualized in 3D-reconstructions of thick filaments from striated muscle. These reconstructions are calculated by averaging methods using images from electron micrographs of grids prepared using numerous filament preparations. Here we propose an alternative method to calculate the 3D-reconstruction of a single thick filament using only a tilt series images recorded by electron tomography. Relaxed thick filaments, prepared from tarantula leg muscle homogenates, were negatively stained. Single-axis tilt series of single isolated thick filaments were obtained with the electron microscope at a low electron dose, and recorded on a CCD camera by electron tomography. An IHRSR 3D-recontruction was calculated from the tilt series images of a single thick filament. The reconstruction was enhanced by including in the search stage dual tilt image segments while only single tilt along the filament axis is usually used, as well as applying a band pass filter just before the back projection. The reconstruction from a single filament has a 40 Å resolution and clearly shows the presence of MIH motifs. In contrast, the electron tomogram 3D-reconstruction of the same thick filament - calculated without any image averaging and/or imposition of helical symmetry - only reveals MIH motifs infrequently. This is - to our knowledge - the first application of the IHRSR method to calculate a 3D reconstruction from tilt series images. This single filament IHRSR reconstruction method (SF-IHRSR) should provide a new tool to assess structural differences between well-ordered thick (or thin) filaments in a grid by recording separately their electron tomograms.
Assuntos
Extremidades/anatomia & histologia , Modelos Moleculares , Músculo Estriado/ultraestrutura , Miosinas/ultraestrutura , Aranhas/anatomia & histologia , Animais , Tomografia com Microscopia Eletrônica , Imageamento Tridimensional , Microscopia EletrônicaRESUMO
Ginseng acidic polysaccharide WGPA isolated from the root of Panax ginseng C. A. Meyer was fractionated into WGPA-A and WGPA-N by anion-exchange chromatography. The antifatigue activity of ginseng acidic polysaccharide WGPA has been reported in our previous research. This present study was designed to identify its active component and elucidate the mechanism for preventing chronic fatigue syndrome (CFS). WGPA, WGPA-A and WGPA-N were orally administered to mice once daily for 15 days. The effects of these compounds on physiological biomarkers of oxidative stress and on the morphology of the mitochondria in striated skeletal muscle were assessed. The results of forced swimming test-induced indicated that WGPA and WGPA-A could lengthen the swimming time, while WGPA-N could not. In addition, malondialdehyde and lactate dehydrogenase levels in serum were enhanced; while those of superoxide dismutase and glutathione peroxidase were lowered. Interestingly, the structural degeneration of mitochondria were all ameliorated. These findings suggested that WGPA-A is the active component of WGPA, it might have potential therapeutic effects for CFS and the oxidative stress might be involved in the pathogenesis. Our results also provided essential data for a better understanding of the antifatigue effects of P. ginseng extracts.
Assuntos
Síndrome de Fadiga Crônica/tratamento farmacológico , Panax/química , Fitoterapia , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Polissacarídeos/farmacologia , Polissacarídeos/uso terapêutico , Animais , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Biomarcadores/sangue , Modelos Animais de Doenças , Fadiga/sangue , Fadiga/tratamento farmacológico , Fadiga/metabolismo , Síndrome de Fadiga Crônica/sangue , Síndrome de Fadiga Crônica/metabolismo , Glutationa Peroxidase/sangue , L-Lactato Desidrogenase/sangue , Masculino , Malondialdeído/sangue , Malondialdeído/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Músculo Estriado/efeitos dos fármacos , Músculo Estriado/metabolismo , Músculo Estriado/ultraestrutura , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/química , Raízes de Plantas/química , Polissacarídeos/isolamento & purificação , Superóxido Dismutase/sangue , NataçãoRESUMO
INTRODUCTION AND HYPOTHESIS: Diabetes mellitus (DM) during pregnancy is associated with high levels of urinary incontinence (UI) and pelvic floor muscle dysfunction. Mild DM can lead to changes in urethral striated muscle and extracellular matrix (ECM) in pregnant rats considering both structures as an entire system responsible for urinary continence. METHODS: Ninety-two female Wistar rats were distributed in four experimental groups: virgin, pregnant, diabetic, and diabetic pregnant. In adult life, parental nondiabetic female rats were mated with nondiabetic male rats to obtain newborns. At the first day of birth, newborns received citrate buffer (nondiabetic group) or streptozotocin 100 mg/kg body weight, subcutaneous route (mild DM group). At day 21 of the pregnancy, the rats were lethally anesthetized and the urethra and vagina were extracted as a unit. Urethral and vaginal sections were cut and analyzed by: (a) cytochemical staining for ECM and muscle structural components, (b) immunohistochemistry to identify fast- and slow-muscle fibers, and (c) transmission electron microscopy for ultrastructural analysis of urethral striated muscle. RESULTS: In comparison with the three control groups, variations in the urethral striated muscle and ECM from diabetic pregnant rats were observed including thinning, atrophy, fibrosis, increased area of blood vessels, mitochondria accumulation, increased lipid droplets, glycogen granules associated with colocalization of fast and slow fibers, and a steady decrease in the proportion of fast to slow fibers. CONCLUSIONS: Mild DM and pregnancy can lead to a time-dependent disorder and tissue remodeling in which the urethral striated muscle and ECM has a fundamental function.
Assuntos
Diabetes Mellitus Experimental/patologia , Matriz Extracelular/ultraestrutura , Músculo Estriado/ultraestrutura , Uretra/patologia , Animais , Atrofia , Vasos Sanguíneos/patologia , Feminino , Fibrose , Glicogênio/ultraestrutura , Lipídeos , Mitocôndrias/patologia , Fibras Musculares de Contração Rápida/ultraestrutura , Fibras Musculares de Contração Lenta/ultraestrutura , Gravidez , Ratos Wistar , Uretra/irrigação sanguíneaRESUMO
The striated muscles of Derocheilocaris typica consist of mononucleated cells, each containing one filament bundle. Large muscles consist of two or more cells adjacent to each other. The mitochondria line up along the filament bundle on one side. The nucleus is situated in the mitochondrial row and has a small cytoplasmic area around it filled with glycogen. The sarcomeres are between 3 and 6 µm long. The Z-line and H band are present. Six thin filaments surround one thick filament. All muscles belong to the phasic type. The tubular system emanates from the ends of the muscle cell and penetrates the whole cell. The tubules are formed as cisterns, which also open at the cell membrane at the level of the I bands. They have sarcoplasmic cisterns on both sides forming a continuous triad system. Partially transformed epidermal cells mediate muscle insertions on the cuticle. Tendons are formed with the transformed epidermal cells being supplemented by fibroblasts forming collagen fibers. Dorsal and ventral abdominal muscles are innervated from the dorso-lateral nerve arising from the nerve chain. Each muscle cell receives one axon, which forms one synapse on the mitochondrial-free side of the muscles. Axons form terminal spines, which make axo-axonal synapses.