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Aging is accompanied by a progressive loss of skeletal muscle mass and strength. The mechanisms underlying this phenomenon are certainly multifactorial and still remain to be fully elucidated. Changes in the cell nucleus structure and function have been considered among the possible contributing causes. This review offers an overview of the current knowledge on skeletal muscle nuclei in aging, focusing on the impairment of nuclear pathways potentially involved in age-related muscle decline. In skeletal muscle two types of cells are present: fiber cells, constituting the contractile muscle mass and containing hundreds of myonuclei, and the satellite cells, i.e., the myogenic mononuclear stem cells occurring at the periphery of the fibers and responsible for muscle growth and repair. Research conducted on different experimental models and with different methodological approaches demonstrated that both the myonuclei and satellite cell nuclei of aged skeletal muscles undergo several structural and molecular alterations, affecting chromatin organization, gene expression, and transcriptional and post-transcriptional activities. These alterations play a key role in the impairment of muscle fiber homeostasis and regeneration, thus contributing to the age-related decrease in skeletal muscle mass and function.
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Núcleo Celular , Músculo Esquelético , Músculo Esquelético/metabolismo , Núcleo Celular/metabolismo , Fibras Musculares Esqueléticas/metabolismoRESUMO
The ex vivo treatment of a limited volume of blood with gaseous oxygen-ozone (O2-O3) mixtures and its rapid reinfusion into the patient is a widespread medical procedure. O3 instantly reacts with the blood's antioxidant systems, disappearing before reinfusion, although the molecules formed act as messengers in the organism, inducing multiple antioxidant and anti-inflammatory responses. An appropriate dose of O3 is obviously essential to ensure both safety and therapeutic efficacy, and in recent years, the low-dose O3 concept has led to a significant reduction in the administered O3 concentrations. However, the molecular events triggered by such low concentrations in the blood still need to be fully elucidated. In this basic study, we analysed the molecular modifications induced ex vivo in sheep blood by 5 and 10 µg O3/mL O2 by means of a powerful metabolomics analysis in association with haemogas, light microscopy and bioanalytical assays. This combined approach revealed increased oxygenation and an increased antioxidant capacity in the O3-treated blood, which accorded with the literature. Moreover, original information was obtained on the impact of these low O3 concentrations on the metabolic pathways of amino acids, carbohydrates, lipids and nucleotides, with the modified metabolites being mostly involved in the preservation of the oxidant-antioxidant balance and in energy production.
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Antioxidantes , Ozônio , Humanos , Animais , Ovinos , Antioxidantes/metabolismo , Ozônio/uso terapêutico , Oxidantes , CarboidratosRESUMO
Introduction: A regular physical training is known to contribute to preserve muscle mass and strength, maintaining structure and function of neural and vascular compartments and preventing muscle insulin resistance and inflammation. However, physical activity is progressively reduced during aging causing mobility limitations and poor quality of life. Although physical exercise for rehabilitation purposes (e.g., after fractures or cardiovascular events) or simply aiming to counteract the development of sarcopenia is frequently advised by physicians, nevertheless few data are available on the targets and the global effects on the muscle organ of adapted exercise especially if started at old age. Methods: To contribute answering this question for medical translational purposes, the proteomic profile of the gastrocnemius muscle was analyzed in 24-month-old mice undergoing adapted physical training on a treadmill for 12 weeks or kept under a sedentary lifestyle condition. Proteomic data were implemented by morphological and morphometrical ultrastructural evaluations. Results and Discussion: Data demonstrate that muscles can respond to adapted physical training started at old age, positively modulating their morphology and the proteomic profile fostering protective and saving mechanisms either involving the extracellular compartment as well as muscle cell components and pathways (i.e., mitochondrial processes, cytoplasmic translation pathways, chaperone-dependent protein refolding, regulation of skeletal muscle contraction). Therefore, this study provides important insights on the targets of adapted physical training, which can be regarded as suitable benchmarks for future in vivo studies further exploring the effects of this type of physical activity by functional/metabolic approaches.
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Platelet-rich plasma (PRP) is gaining more and more attention in regenerative medicine as an innovative and efficient therapeutic approach. The regenerative properties of PRP rely on the numerous bioactive molecules released by the platelets: growth factors are involved in proliferation and differentiation of endothelial cells and fibroblasts, angiogenesis and extracellular matrix formation, while cytokines are mainly involved in immune cell recruitment and inflammation modulation. Attempts are ongoing to improve the therapeutic potential of PRP by combining it with agents able to promote regenerative processes. Two interesting candidates are ozone, administered at low doses as gaseous oxygen-ozone mixtures, and procaine. In the present study, we investigated the effects induced on platelets by the in vitro treatment of PRP with ozone or procaine, or both. We combined transmission electron microscopy to obtain information on platelet modifications and bioanalytical assays to quantify the secreted factors. The results demonstrate that, although platelets were already activated by the procedure to prepare PRP, both ozone and procaine induced differential morpho-functional modifications in platelets resulting in an increased release of factors. In detail, ozone induced an increase in surface protrusions and open canalicular system dilation suggestive of a marked α-granule release, while procaine caused a decrease in surface protrusions and open canalicular system dilation but a remarkable increase in microvesicle release suggestive of high secretory activity. Consistently, nine of the thirteen platelet-derived factors analysed in the PRP serum significantly increased after treatment with ozone and/or procaine. Therefore, ozone and procaine proved to have a remarkable stimulating potential without causing any damage to platelets, probably because they act through physiological, although different, secretory pathways.
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Ozônio , Plasma Rico em Plaquetas , Ozônio/farmacologia , Procaína/farmacologia , Procaína/metabolismo , Células Endoteliais , Citocinas/metabolismo , Plasma Rico em Plaquetas/metabolismoRESUMO
Missense mutations in MYOT encoding the sarcomeric Z-disk protein myotilin cause three main myopathic phenotypes including proximal limb-girdle muscular dystrophy, spheroid body myopathy, and late-onset distal myopathy. We describe a family carrying a heterozygous MYOT deletion (Tyr4_His9del) that clinically was characterized by an early-adult onset distal muscle weakness and pathologically by a myofibrillar myopathy (MFM). Molecular modeling of the full-length myotilin protein revealed that the 4-YERPKH-9 amino acids are involved in local interactions within the N-terminal portion of myotilin. Injection of in vitro synthetized mutated human MYOT RNA or of plasmid carrying its cDNA sequence in zebrafish embryos led to muscle defects characterized by sarcomeric disorganization of muscle fibers and widening of the I-band, and severe motor impairments. We identify MYOT novel Tyr4_His9 deletion as the cause of an early-onset MFM with a distal myopathy phenotype and provide data supporting the importance of the amino acid sequence for the structural role of myotilin in the sarcomeric organization of myofibers.
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Miopatias Distais , Proteínas Musculares , Adulto , Animais , Humanos , Conectina/genética , Proteínas dos Microfilamentos/genética , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Mutação , Peixe-ZebraRESUMO
Myofibrillar myopathies (MFMs) are a group of hereditary neuromuscular disorders sharing common histological features, such as myofibrillar derangement, Z-disk disintegration, and the accumulation of degradation products into protein aggregates. They are caused by mutations in several genes that encode either structural proteins or molecular chaperones. Nevertheless, the mechanisms by which mutated genes result in protein aggregation are still unknown. To unveil the role of myotilin and αB-crystallin in the pathogenesis of MFM, we injected zebrafish fertilized eggs at the one-cell stage with expression plasmids harboring cDNA sequences of human wildtype or mutated MYOT (p.Ser95Ile) and human wildtype or mutated CRYAB (p.Gly154Ser). We evaluated the effects on fish survival, motor behavior, muscle structure and development. We found that transgenic zebrafish showed morphological defects that were more severe in those overexpressing mutant genes. which developed a myopathic phenotype consistent with that of human myofibrillar myopathy, including the formation of protein aggregates. Results indicate that pathogenic mutations in myotilin and αB-crystallin genes associated with MFM cause a structural and functional impairment of the skeletal muscle in zebrafish, thereby making this non-mammalian organism a powerful model to dissect disease pathogenesis and find possible druggable targets.
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Cristalinas , Miopatias Congênitas Estruturais , Animais , Humanos , Cadeia B de alfa-Cristalina/genética , Cadeia B de alfa-Cristalina/metabolismo , Cristalinas/genética , Músculo Esquelético/patologia , Mutação , Miofibrilas/metabolismo , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/metabolismo , Agregados Proteicos , Peixe-Zebra/genéticaRESUMO
Down syndrome (DS) is a genetically based disease caused by triplication of chromosome 21. DS is characterized by multi-systemic premature aging associated with deficit in motor coordination, balance, and postural control. Using a morphological, morphometrical, and immunocytochemical ultrastructural approach, this study investigated in vastus lateralis muscle of Ts65Dn mouse, a murine model of DS, the effect of an adapted physical training on the extracellular matrix (ECM) characteristics and whether the forecasted exercise-induced ECM remodeling impacts on sarcomere organization. Morphometry demonstrated thicker basement membrane and larger collagen bundles with larger interfibrillar spacing as well as irregularly arrayed myofibrils and lower telethonin density on Z-lines in trisomic versus euploid sedentary mice. In agreement with the multi-systemic premature aging described in DS, these ECM alterations were similar to those previously observed in skeletal muscle of aged mice. Adapted physical training induced remodeling of ECM in both trisomic and euploid mice, that is, enlargement of the collagen bundles associated with hypertrophy of collagen fibrils and reduction of the interfibrillar spacing. A re-alignment of the myofibrils and a higher telethonin density on Z-line was found in trisomic mice. Altogether, our findings suggest that physical training is an effective tool in limiting/counteracting the trisomy-associated musculoskeletal structural anomalies. The current findings constitute a solid experimental background for further study investigating the possible positive effect of physical training on skeletal muscle performance. RESEARCH HIGHLIGHTS: Vastus lateralis muscle of trisomic mice shows aging-like alterations of extracellular matrix. Training promotes extracellular matrix remodeling. Training may be an effective tool to counteract trisomy-associated alterations of skeletal muscle.
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Senilidade Prematura , Síndrome de Down , Camundongos , Animais , Síndrome de Down/genética , Trissomia , Modelos Animais de Doenças , Matriz Extracelular , Colágeno , Músculo EsqueléticoRESUMO
Down syndrome (DS) is a genetically-based disease based on the trisomy of chromosome 21 (Hsa21). DS is characterized by intellectual disability in association with several pathological traits among which early aging and altered motor coordination are prominent. Physical training or passive exercise were found to be useful in counteracting motor impairment in DS subjects. In this study we used the Ts65Dn mouse, a widely accepted animal model of DS, to investigate the ultrastructural architecture of the medullary motor neuron cell nucleus taken as marker of the cell functional state. Using transmission electron microscopy, ultrastructural morphometry, and immunocytochemistry we carried out a detailed investigation of possible trisomy-related alteration(s) of nuclear constituents, which are known to vary their amount and distribution as a function of nuclear activity, as well as the effect of adapted physical training upon them. Results demonstrated that trisomy per se affects nuclear constituents to a limited extent; however, adapted physical training is able to chronically stimulate pre-mRNA transcription and processing activity in motor neuron nuclei of trisomic mice, although to a lesser extent than in their euploid mates. These findings are a step towards understanding the mechanisms underlying the positive effect of physical activity in DS.
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Síndrome de Down , Camundongos , Animais , Síndrome de Down/genética , Trissomia , Neurônios Motores/patologia , Núcleo Celular/patologia , Exercício FísicoRESUMO
The mild oxidative stress induced by low doses of gaseous ozone (O3) activates the antioxidant cell response through the nuclear factor erythroid 2-related factor 2 (Nrf2), thus inducing beneficial effects without cell damage. Mitochondria are sensitive to mild oxidative stress and represent a susceptible O3 target. In this in vitro study, we investigated the mitochondrial response to low O3 doses in the immortalized, non-tumoral muscle C2C12 cells; a multimodal approach including fluorescence microscopy, transmission electron microscopy and biochemistry was used. Results demonstrated that mitochondrial features are finely tuned by low O3 doses. The O3 concentration of 10 µg maintained normal levels of mitochondria-associated Nrf2, promoted the mitochondrial increase of size and cristae extension, reduced cellular reactive oxygen species (ROS) and prevented cell death. Conversely, in 20 µg O3-treated cells, where the association of Nrf2 with the mitochondria drastically dropped, mitochondria underwent more significant swelling, and ROS and cell death increased. This study, therefore, adds original evidence for the involvement of Nrf2 in the dose-dependent response to low O3 concentrations not only as an Antioxidant Response Elements (ARE) gene activator but also as a regulatory/protective factor of mitochondrial function.
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Ozônio , Camundongos , Animais , Espécies Reativas de Oxigênio/metabolismo , Ozônio/farmacologia , Ozônio/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Estresse Oxidativo , Mioblastos/metabolismo , Mitocôndrias/metabolismoRESUMO
Uranyl acetate solution has widely been used as staining reagent for samples processed for ultrastructural morphology, cytochemistry, and immunocytochemistry. Although uranyl acetate guarantees high performance as a staining reagent, the radioactive uranyl salts make its use and purchase severely restricted. In this view, we used a non-radioactive lanthanide mix solution as contrasting dye for both nucleoplasmic and nucleolar ribonucleoprotein-containing components. This method guarantees a good contrast without masking the probe-antigen immunoreaction, thus proving to be a suitable tool for high-resolution studies of both cyto- and immunocytochemistry on acrylic resin-embedded samples.
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Elementos da Série dos Lantanídeos , Sais , Resinas Acrílicas , Microscopia Eletrônica de Transmissão , Compostos Organometálicos , Ribonucleoproteínas , Coloração e RotulagemRESUMO
As a complementary, adjuvant or palliative cure, ozone therapy has increasingly been used globally on a wide variety of diseases [...].
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Ozônio , Ozônio/uso terapêuticoRESUMO
Natively monomeric RNase A can oligomerize upon lyophilization from 40% acetic acid solutions or when it is heated at high concentrations in various solvents. In this way, it produces many dimeric or oligomeric conformers through the three-dimensional domain swapping (3D-DS) mechanism involving both RNase A N- or/and C-termini. Here, we found many of these oligomers evolving toward not negligible amounts of large derivatives after being stored for up to 15 months at 4 °C in phosphate buffer. We call these species super-aggregates (SAs). Notably, SAs do not originate from native RNase A monomer or from oligomers characterized by the exclusive presence of the C-terminus swapping of the enzyme subunits as well. Instead, the swapping of at least two subunits' N-termini is mandatory to produce them. Through immunoblotting, SAs are confirmed to derive from RNase A even if they retain only low ribonucleolytic activity. Then, their interaction registered with Thioflavin-T (ThT), in addition to TEM analyses, indicate SAs are large and circular but not "amyloid-like" derivatives. This confirms that RNase A acts as an "auto-chaperone", although it displays many amyloid-prone short segments, including the 16-22 loop included in its N-terminus. Therefore, we hypothesize the opening of RNase A N-terminus, and hence its oligomerization through 3D-DS, may represent a preliminary step favoring massive RNase A aggregation. Interestingly, this process is slow and requires low temperatures to limit the concomitant oligomers' dissociation to the native monomer. These data and the hypothesis proposed are discussed in the light of protein aggregation in general, and of possible future applications to contrast amyloidosis.
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Amiloidose , Ribonuclease Pancreático , Acetatos , Amiloide , Endorribonucleases/metabolismo , Humanos , Fosfatos , Agregados Proteicos , Estrutura Terciária de Proteína , Ribonuclease Pancreático/metabolismo , Ribonucleases/metabolismo , SolventesRESUMO
Oxygen-ozone (O2 -O3 ) therapy is an adjuvant/complementary treatment based on the activation of antioxidant and cytoprotective pathways driven by the nuclear factor erythroid 2-related factor 2 (Nrf2). Many drugs, including dimethyl fumarate (DMF), that are used to reduce inflammation in oxidative-stress-related neurodegenerative diseases, act through the Nrf2-pathway. The scope of the present investigation was to get a deeper insight into the mechanisms responsible for the beneficial result of O2 -O3 treatment in some neurodegenerative diseases. To do this, we used an integrated approach of multimodal microscopy (bright-field and fluorescence microscopy, transmission and scanning electron microscopy) and biomolecular techniques to investigate the effects of the low O3 concentrations currently used in clinical practice in lipopolysaccharide (LPS)-activated microglial cells human microglial clone 3 (HMC3) and in DMF-treated LPS-activated (LPS + DMF) HMC3 cells. The results at light and electron microscopy showed that LPS-activation induced morphological modifications of HMC3 cells from elongated/branched to larger roundish shape, cytoplasmic accumulation of lipid droplets, decreased electron density of the cytoplasm and mitochondria, decreased amount of Nrf2 and increased migration rate, while biomolecular data demonstrated that Heme oxygenase 1 gene expression and the secretion of the pro-inflammatory cytokines, Interleukin-6, and tumor necrosis factor-α augmented. O3 treatment did not affect cell viability, proliferation, and morphological features of both LPS-activated and LPS + DMF cells, whereas the cell motility and the secretion of pro-inflammatory cytokines were significantly decreased. This evidence suggests that modulation of microglia activity may contribute to the beneficial effects of the O2 -O3 therapy in patients with neurodegenerative disorders characterized by chronic inflammation. HIGHLIGHTS: Low-dose ozone (O3 ) does not damage activated microglial cells in vitro Low-dose O3 decreases cell motility and pro-inflammatory cytokine secretion in activated microglial cells in vitro Low-dose O3 potentiates the effect of an anti-inflammatory drug on activated microglial cells.
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Doenças Neurodegenerativas , Ozônio , Humanos , Microglia/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/farmacologia , Fator 2 Relacionado a NF-E2/uso terapêutico , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/uso terapêutico , Ozônio/farmacologia , Ozônio/metabolismo , Ozônio/uso terapêutico , Microscopia , Inflamação/tratamento farmacológico , Citocinas , Fumarato de Dimetilo/metabolismo , Fumarato de Dimetilo/farmacologia , Fumarato de Dimetilo/uso terapêuticoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer with an overall 5-year survival rate of less than 9%. The high aggressiveness of PDAC is linked to the presence of a subpopulation of cancer cells with a greater tumorigenic capacity, generically called cancer stem cells (CSCs). CSCs present a heterogeneous metabolic profile that might be supported by an adaptation of mitochondrial function; however, the role of this organelle in the development and maintenance of CSCs remains controversial. To determine the role of mitochondria in CSCs over longer periods, which may reflect more accurately their quiescent state, we studied the mitochondrial physiology in CSCs at short-, medium-, and long-term culture periods. We found that CSCs show a significant increase in mitochondrial mass, more mitochondrial fusion, and higher mRNA expression of genes involved in mitochondrial biogenesis than parental cells. These changes are accompanied by a regulation of the activities of OXPHOS complexes II and IV. Furthermore, the protein OPA1, which is involved in mitochondrial dynamics, is overexpressed in CSCs and modulates the tumorsphere formation. Our findings indicate that CSCs undergo mitochondrial remodeling during the stemness acquisition process, which could be exploited as a promising therapeutic target against pancreatic CSCs.
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Two likely causative mutations in the RYR1 gene were identified in two patients with myopathy with tubular aggregates, but no evidence of cores or core-like pathology on muscle biopsy. These patients were clinically evaluated and underwent routine laboratory investigations, electrophysiologic tests, muscle biopsy and muscle magnetic resonance imaging (MRI). They reported stiffness of the muscles following sustained activity or cold exposure and had serum creatine kinase elevation. The identified RYR1 mutations (p.Thr2206Met or p.Gly2434Arg, in patient 1 and patient 2, respectively) were previously identified in individuals with malignant hyperthermia susceptibility and are reported as causative according to the European Malignant Hyperthermia Group rules. To our knowledge, these data represent the first identification of causative mutations in the RYR1 gene in patients with tubular aggregate myopathy and extend the spectrum of histological alterations caused by mutation in the RYR1 gene.
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Hipertermia Maligna , Miopatias Congênitas Estruturais , Humanos , Hipertermia Maligna/genética , Hipertermia Maligna/patologia , Músculo Esquelético/patologia , Mutação/genética , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/patologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genéticaRESUMO
Down syndrome (DS) is characterized by muscle hypotonia and low muscle strength associated with motor dysfunction. Elucidation of the determinants of muscle weakness in DS would be relevant for therapeutic approaches aimed at treating/mitigating a physical disability with a strong impact on the quality of life in persons with DS. The Ts65Dn mice is a recognized mouse model of DS, with trisomic mice presenting gross motor and muscle phenotypes. The aim of this work was to assess the effect of physical exercise, a well-known tool to improve skeletal muscle condition, in the hindlimbs of trisomic and euploid male mice using quantitative magnetic resonance imaging (MRI). Magnetic resonance spectroscopy (MRS) metabolomics and histological fiber typing were used to further characterize the post-exercise muscle. Quantitative MRI showed not significantly different amounts of skeletal muscle in proximal hindlimbs in trisomic and euploid mice both at baseline and after physical exercise (P>0.05). Similar results were obtained for hindlimbs subfascia adipose tissue, and subcutaneous adipose tissue (P>0.05). MRS showed lower amounts of exercise-related metabolites (valine, isoleucine, leucine) in euploid vs. trisomic mice after exercise (P≤0.05). The percentage of slow-twitch fibers was similar in the two genotypes (P>0.05). We conclude that in DS adapted physical exercise (one month of training) does not induce quantitative changes in skeletal muscle or fiber type composition therein; however, the metabolic response of skeletal muscle to exercise may be affected by trisomy. These findings prompt further research investigating the role of physical exercise as a cue to clarify the mechanisms of the muscular deficit found in DS.
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BACKGROUND AND OBJECTIVE: The skeletal muscle is composed of integrated tissues mainly composed of myofibers i.e., long, cylindrical syncytia, whose cytoplasm is mostly occupied by parallel myofibrils. In section, each myofibril is organized in serially end-to-end arranged sarcomeres connected by Z lines. In muscle disorders, these structural and functional units can undergo structural alterations in terms of Z-line and sarcomere lengths, as well as lateral alignment of Z-line among adjacent myofibrils. In this view, objectifying alterations of the myofibril and sarcomere architecture would provide a solid foundation for qualitative observations. In this work, specific quantitative parameters characterizing the sarcomere and myofibril arrangement were defined using a computerized analysis of ultrastructural images. METHODS: computerized analysis was carried out on transmission electron microscopy pictures of the murine vastus lateralis muscle. Samples from both euploid (control) and trisomic (showing myofiber alterations) Ts65Dn mice were used. Two routines were written in MATLAB to measure specific structural parameters on sarcomeres and myofibrils. The output included the Z-line, M-line, and sarcomere lengths, the Aspect Ratio (AsR) and Curviness (Cur) sarcomere shape parameters, myofibril axis (α angle), and the H parameter (evaluation of sequence of Z-lines of adjacent myofibrils). RESULTS: Both routines worked well in control (euploid) skeletal muscle yielding consistent quantitative data of sarcomere and myofibril structural organization. In comparison with euploid, trisomic muscle showed statistically significant lower Z-line length, similar M-line length, and statistically significant lower sarcomere length. Both AsR and Cur were statistically significantly lower in trisomic muscle, suggesting the sarcomere is barrel-shaped in the latter. The angle (α) distribution showed that the sarcomere axes are almost parallel in euploid muscle, while a large variability occurs in trisomic tissue. The mean value of H was significantly higher in trisomic versus euploid muscle indicating that Z-lines are not perfectly aligned in trisomic muscle. CONCLUSIONS: Our procedure allowed us to accurately extract and quantify sarcomere and myofibril parameters from the high-resolution electron micrographs thereby yielding an effective tool to quantitatively define trisomy-associated muscle alterations. These results pave the way to future objective quantification of skeletal muscle changes in pathological conditions. SHORT ABSTRACT: The skeletal muscle is composed of integrated tissues mainly composed of myofibers i.e., long, cylindrical syncytia, whose cytoplasm is mostly occupied by parallel myofibrils organized in serially end-to-end arranged sarcomeres. Several pieces of evidence have highlighted that in muscle disorders and diseases the sarcomere structure may be altered. Therefore, objectifying alterations of the myofibril and sarcomere architecture would provide a solid foundation for qualitative observations. A computerized analysis was carried out on transmission electron microscopy images of euploid (control) and trisomic (showing myofiber alterations) skeletal muscle. Two routines were written in MATLAB to measure nine sarcomere and myofibril structural parameters. Our computational method confirmed and expanded on previous qualitative ultrastructural findings defining several trisomy-associated skeletal muscle alterations. The proposed procedure is a potentially useful tool to quantitatively define skeletal muscle changes in pathological conditions involving the sarcomere.
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Miofibrilas , Sarcômeros , Animais , Camundongos , Microscopia Eletrônica de Transmissão , Músculo Esquelético/diagnóstico por imagemRESUMO
Aging is characterized by a progressive decline of skeletal muscle (SM) mass and strength which may lead to sarcopenia in older persons. To date, a limited number of studies have been performed in the old SM looking at the whole, complex network of the extracellular matrix (i.e., matrisome) and its aging-associated changes. In this study, skeletal muscle proteins were isolated from whole gastrocnemius muscles of adult (12 mo.) and old (24 mo.) mice using three sequential extractions, each one analyzed by liquid chromatography with tandem mass spectrometry. Muscle sections were investigated using fluorescence- and transmission electron microscopy. This study provided the first characterization of the matrisome in the old SM demonstrating several statistically significantly increased matrisome proteins in the old vs. adult SM. Several proteomic findings were confirmed and expanded by morphological data. The current findings shed new light on the mutually cooperative interplay between cells and the extracellular environment in the aging SM. These data open the door for a better understanding of the mechanisms modulating myocellular behavior in aging (e.g., by altering mechano-sensing stimuli as well as signaling pathways) and their contribution to age-dependent muscle dysfunction.
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Envelhecimento/metabolismo , Matriz Extracelular/metabolismo , Músculo Esquelético/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Fatores Etários , Animais , Cromatografia Líquida , Colágeno/metabolismo , Laminina/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Músculo Esquelético/ultraestrutura , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/metabolismo , Espectrometria de Massas em TandemRESUMO
Oxygen-ozone (O2-O3) therapy is increasingly applied as a complementary/adjuvant treatment for several diseases; however, the biological mechanisms accounting for the efficacy of low O3 concentrations need further investigations to understand the possibly multiple effects on the different cell types. In this work, we focused our attention on fibroblasts as ubiquitous connective cells playing roles in the body architecture, in the homeostasis of tissue-resident cells, and in many physiological and pathological processes. Using an established human fibroblast cell line as an in vitro model, we adopted a multimodal approach to explore a panel of cell structural and functional features, combining light and electron microscopy, Western blot analysis, real-time quantitative polymerase chain reaction, and multiplex assays for cytokines. The administration of O2-O3 gas mixtures induced multiple effects on fibroblasts, depending on their activation state: in non-activated fibroblasts, O3 stimulated proliferation, formation of cell surface protrusions, antioxidant response, and IL-6 and TGF-ß1 secretion, while in LPS-activated fibroblasts, O3 stimulated only antioxidant response and cytokines secretion. Therefore, the low O3 concentrations used in this study induced activation-like responses in non-activated fibroblasts, whereas in already activated fibroblasts, the cell protective capability was potentiated.
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Fibroblastos/efeitos dos fármacos , Oxidantes Fotoquímicos/farmacologia , Ozônio/farmacologia , Linhagem Celular , Proliferação de Células , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Fibroblastos/ultraestrutura , Heme Oxigenase-1/metabolismo , Humanos , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Fator de Crescimento Transformador beta/metabolismoRESUMO
During the last few years, for therapeutic purposes in oncology, considerable attention has been focused on a method called magnetic fluid hyperthermia (MFH) based on local heating of tumor cells. In this paper, an innovative, promising nanomaterial, M48 composed of iron oxide-based phases has been tested. M48 shows self-regulating temperature due to the observable second order magnetic phase transition from ferromagnetic to paramagnetic state. A specific hydrophilic coating based on both citrate ions and glucose molecules allows high biocompatibility of the nanomaterial in biological matrices and its use in vivo. MFH mediator efficiency is demonstrated in vitro and in vivo in breast cancer cells and tumors, confirming excellent features for biomedical application. The temperature increase, up to the Curie temperature, gives rise to a phase transition from ferromagnetic to paramagnetic state, promoting a shortage of the r2 transversal relaxivity that allows a switch in the contrast in Magnetic Resonance Imaging (MRI). Combining this feature with a competitive high transversal (spin-spin) relaxivity, M48 paves the way for a new class of temperature sensitive T2 relaxing contrast agents. Overall, the results obtained in this study prepare for a more affordable and tunable heating mechanism preventing the damages of the surrounding healthy tissues and, at the same time, allowing monitoring of the temperature reached.