RESUMO
Hamstring muscle injuries are the most prevalent among athletes who engage in sprinting activities. Their most frequent location is where the long head of the biceps femoris joins with the semitendinosus muscle to form the conjoint hamstring tendon. Just distal to this area, an additional group of fibers of the semitendinosus originate from medial aspect of biceps femoris. The objective of this study was to analyze the morphological characteristics of this union and to discuss its potential role in hamstring tears. Anatomical dissection was performed on 35 thighs. Samples obtained from this region were sectioned and stained with Masson's trichrome for further histological evaluation. A group of fibers from the semitendinosus muscle originating from the long head of the biceps femoris were observed in all 35 specimens. This origin was located 67 ± 12 mm from the ischial tuberosity and was 32 ± 14 mm in length. This group of muscle fibers had a width of 10.9 ± 5.3 mm and a thickness in the anteroposterior axis of 3.2 ± 1.4 mm. Its pennation angle was 9.2 ± 1.5 degrees. Microscopic examination showed muscle cells from both muscles contacting interposed tendinous tissue. In conclusion, fibers of the semitendinosus muscle consistently arise from the proximal aspect of the long head of biceps femoris. The morphological characteristics of this junction have functional implications. The horizontal component of the semitendinosus vector could pull the long head of the biceps femoris medially during its shortening-lengthening cycle, rendering it an intrinsic risk factor for hamstring injuries.
Assuntos
Músculos Isquiossurais/anatomia & histologia , Idoso , Variação Anatômica , Traumatismos em Atletas/patologia , Cadáver , Dissecação/métodos , Feminino , Músculos Isquiossurais/lesões , Humanos , Masculino , Fibras Musculares Esqueléticas/citologia , Fatores de Risco , Tendões/anatomia & histologiaRESUMO
Zika virus (ZIKV) infection became a worldwide concern due to its correlation with the development of microcephaly and other neurological disorders. ZIKV neurotropism is well characterized, but the role of peripheral viral amplification to brain infection remains unknown. Here, we found that ZIKV replicates in human primary skeletal muscle myoblasts, impairing its differentiation into myotubes but not interfering with the integrity of the already-formed muscle fibers. Using mouse models, we showed ZIKV tropism to muscle tissue either during embryogenesis after maternal transmission or when infection occurred after birth. Interestingly, ZIKV replication in the mouse skeletal muscle started immediately after ZIKV inoculation, preceding viral RNA detection in the brain and causing no disruption to the integrity of the blood brain barrier, and remained active for more than 2 weeks, whereas replication in the spleen and liver were not sustained over time. In addition, ZIKV infection of the skeletal muscle induces necrotic lesions, inflammation, and fiber atrophy. We also found a reduction in the expression of regulatory myogenic factors that are essential for muscle repair after injury. Taken together, our results indicate that the skeletal muscle is an early site of viral amplification and lesion that may result in late consequences in muscle development after ZIKV infection. IMPORTANCE Zika Virus (ZIKV) neurotropism and its deleterious effects on central nervous system have been well characterized. However, investigations of the initial replication sites for the establishment of infection and viral spread to neural tissues remain underexplored. A complete description of the range of ZIKV-induced lesions and others factors that can influence the severity of the disease is necessary to prevent ZIKV's deleterious effects. ZIKV has been shown to access the central nervous system without significantly affecting blood-brain barrier permeability. Here, we demonstrated that skeletal muscle is an earlier site of ZIKV replication, contributing to the increase of peripheral ZIKV load. ZIKV replication in muscle promotes necrotic lesions and inflammation and also impairs myogenesis. Overall, our findings showed that skeletal muscle is involved in pathogenesis and opens new fields in the investigation of the long-term consequences of early infection.
Assuntos
Fibras Musculares Esqueléticas/virologia , Infecção por Zika virus/virologia , Zika virus/fisiologia , Aedes , Animais , Animais Recém-Nascidos , Linhagem Celular , Feminino , Humanos , Transmissão Vertical de Doenças Infecciosas , Camundongos , Camundongos Knockout , Fibras Musculares Esqueléticas/citologia , Mioblastos , Replicação ViralRESUMO
The myotendinous junction (MTJ) is the muscle-tendon interface and constitutes an integrated mechanical unit to force transmission. Joint immobilization promotes muscle atrophy via disuse, while physical exercise can be used as an adaptative stimulus. In this study, we aimed to investigate the components of the MTJ and their adaptations and the associated elements triggered with aquatic training after joint immobilization. Forty-four male Wistar rats were divided into sedentary (SD), aquatic training (AT), immobilization (IM), and immobilization/aquatic training (IMAT) groups. The samples were processed to measure fiber area, nuclear fractal dimension, MTJ nuclear density, identification of telocytes, sarcomeres, and MTJ perimeter length. In the AT group, the maintenance of ultrastructure and elements in the MTJ region were observed; the IM group presented muscle atrophy effects with reduced MTJ perimeter; the IMAT group demonstrated that aquatic training after joint immobilization promotes benefits in the muscle fiber area and fractal dimension, in the MTJ region shows longer sarcomeres and MTJ perimeter. We identified the presence of telocytes in the MTJ region in all experimental groups. We concluded that aquatic training is an effective rehabilitation method after joint immobilization due to reduced muscle atrophy and regeneration effects on MTJ in rats.
Assuntos
Adaptação Fisiológica , Imobilização , Articulações , Condicionamento Físico Animal , Esforço Físico , Tendões/fisiologia , Animais , Masculino , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestrutura , Ratos , Sarcômeros/ultraestrutura , Tendões/citologia , Tendões/ultraestruturaRESUMO
Skeletal muscle has the remarkable ability to regenerate. However, with age and disease muscle strength and function decline. Myofiber size, which is affected by injury and disease, is a critical measurement to assess muscle health. Here, we test and apply Cellpose, a recently developed deep learning algorithm, to automatically segment myofibers within murine skeletal muscle. We first show that tissue fixation is necessary to preserve cellular structures such as primary cilia, small cellular antennae, and adipocyte lipid droplets. However, fixation generates heterogeneous myofiber labeling, which impedes intensity-based segmentation. We demonstrate that Cellpose efficiently delineates thousands of individual myofibers outlined by a variety of markers, even within fixed tissue with highly uneven myofiber staining. We created a novel ImageJ plugin (LabelsToRois) that allows processing of multiple Cellpose segmentation images in batch. The plugin also contains a semi-automatic erosion function to correct for the area bias introduced by the different stainings, thereby identifying myofibers as accurately as human experts. We successfully applied our segmentation pipeline to uncover myofiber regeneration differences between two different muscle injury models, cardiotoxin and glycerol. Thus, Cellpose combined with LabelsToRois allows for fast, unbiased, and reproducible myofiber quantification for a variety of staining and fixation conditions.
Assuntos
Histocitoquímica , Processamento de Imagem Assistida por Computador , Microscopia , Fibras Musculares Esqueléticas/citologia , Músculo Esquelético/citologia , Algoritmos , Animais , Biologia Computacional/métodos , Histocitoquímica/métodos , Processamento de Imagem Assistida por Computador/métodos , Camundongos , Microscopia/métodos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , SoftwareRESUMO
Isolated myofibers are commonly used to understand the function of skeletal muscle in vivo. This can involve single isolated myofibers obtained from dissection or from enzymatic dissociation. Isolation via dissection allows control of sarcomere length and preserves tendon attachment but is labor-intensive, time-consuming and yields few viable myofibers. In contrast, enzymatic dissociation is fast and facile, produces hundreds of myofibers, and more importantly reduces the number of muscles/animals needed for studies. Biomechanical properties of the sarcolemma have been studied using myofibers from the extensor digitorum longus, but this has been limited to dissected myofibers, making data collection slow and difficult. We have modified this tool to perform biomechanical measurements of the sarcolemma in dissociated myofibers from the flexor digitorum brevis.
Assuntos
Técnicas de Cultura de Células/métodos , Fibras Musculares Esqueléticas/citologia , Sarcolema/fisiologia , Animais , Fenômenos Biomecânicos , Elasticidade , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Fibras Musculares Esqueléticas/ultraestruturaRESUMO
Satellite cells (SCs) are myogenic progenitors responsible for skeletal muscle regeneration and maintenance. Upon activation, SCs enter a phase of robust proliferation followed by terminal differentiation. Underlying this myogenic progression, the sequential expression of muscle regulatory transcription factors (MRFs) and the downregulation of transcription factor paired box gene 7 (Pax7) are key steps regulating SC fate. In addition to transcriptional regulation, post-translational control of Pax7 and the MRFs provides another layer of spatiotemporal control to the myogenic process. In this context, previous work showed that Pax7 is ubiquitinated by the E3 ligase neural precursor cell-expressed developmentally downregulated protein 4 and interacts with several proteins related to the ubiquitin-proteasome system, including the deubiquitinase ubiquitin-specific protease 7 (USP7). Although USP7 functions in diverse cellular contexts, its role(s) during myogenesis remains poorly explored. Here, we show that USP7 is transiently expressed in adult muscle progenitors, correlating with the onset of myogenin expression, while it is downregulated in newly formed myotubes/myofibers. Acute inhibition of USP7 activity upon muscle injury results in persistent expression of early regeneration markers and a significant reduction in the diameter of regenerating myofibers. At the molecular level, USP7 downregulation or pharmacological inhibition impairs muscle differentiation by affecting myogenin stability. Co-immunoprecipitation and in vitro activity assays indicate that myogenin is a novel USP7 target for deubiquitination. These results suggest that USP7 regulates SC myogenic progression by enhancing myogenin stability.
Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Desenvolvimento Muscular/genética , Miogenina/genética , Células Satélites de Músculo Esquelético/metabolismo , Peptidase 7 Específica de Ubiquitina/genética , Animais , Linhagem Celular , Proliferação de Células/genética , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Miogenina/metabolismo , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Estabilidade Proteica , Regeneração/genética , Células Satélites de Músculo Esquelético/citologia , Peptidase 7 Específica de Ubiquitina/metabolismoRESUMO
Several skeletal muscle diseases are characterized by fibrosis, the excessive accumulation of extracellular matrix. Transforming growth factor-ß (TGF-ß) and connective tissue growth factor (CCN2/CTGF) are two profibrotic factors augmented in fibrotic skeletal muscle, together with signs of reduced vasculature that implies a decrease in oxygen supply. We observed that fibrotic muscles are characterized by the presence of positive nuclei for hypoxia-inducible factor-1α (HIF-1α), a key mediator of the hypoxia response. However, it is not clear how a hypoxic environment could contribute to the fibrotic phenotype in skeletal muscle. We evaluated the role of hypoxia and TGF-ß on CCN2 expression in vitro. Fibroblasts, myoblasts and differentiated myotubes were incubated with TGF-ß1 under hypoxic conditions. Hypoxia and TGF-ß1 induced CCN2 expression synergistically in myotubes but not in fibroblasts or undifferentiated muscle progenitors. This induction requires HIF-1α and the Smad-independent TGF-ß signaling pathway. We performed in vivo experiments using pharmacological stabilization of HIF-1α or hypoxia-induced via hindlimb ischemia together with intramuscular injections of TGF-ß1, and we found increased CCN2 expression. These observations suggest that hypoxic signaling together with TGF-ß signaling, which are both characteristics of a fibrotic skeletal muscle environment, induce the expression of CCN2 in skeletal muscle fibers and myotubes.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Músculo Esquelético/patologia , Fator de Crescimento Transformador beta1/administração & dosagem , Regulação para Cima , Animais , Diferenciação Celular , Hipóxia Celular , Linhagem Celular , Modelos Animais de Doenças , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose , Injeções Intramusculares , Isquemia/etiologia , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Células NIH 3T3 , Transdução de Sinais , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
The bioactive peptide bradykinin obtained from cleavage of precursor kininogens activates the kinin-B2 receptor functioning in induction of inflammation and vasodilatation. In addition, bradykinin participates in kidney and cardiovascular development and neuronal and muscle differentiation. Here we show that kinin-B2 receptors are expressed throughout differentiation of murine C2C12 myoblasts into myotubes. An autocrine loop between receptor activation and bradykinin secretion is suggested, since bradykinin secretion is significantly reduced in the presence of the kinin-B2 receptor antagonist HOE-140 during differentiation. Expression of skeletal muscle markers and regenerative capacity were decreased after pharmacological inhibition or genetic ablation of the B2 receptor, while its antagonism increased the number of myoblasts in culture. In summary, the present work reveals to date no functions described for the B2 receptor in muscle regeneration due to the control of proliferation and differentiation of muscle precursor cells.
Assuntos
Diferenciação Celular , Músculo Esquelético/fisiologia , Mioblastos/citologia , Receptor B2 da Bradicinina/metabolismo , Regeneração , Animais , Biomarcadores/metabolismo , Bradicinina/metabolismo , Cardiotoxinas/administração & dosagem , Linhagem Celular , Proliferação de Células , Citoesqueleto/metabolismo , Deleção de Genes , Cininogênios/genética , Cininogênios/metabolismo , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Cadeias Pesadas de Miosina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor B2 da Bradicinina/genéticaRESUMO
Low-level laser irradiation (LLLI) has been used as a non-invasive method to improve muscular regeneration capability. However, the molecular mechanisms by which LLLI exerts these effects remain largely unknown. Here, we described global gene expression profiling analysis in C2C12 myoblasts after LLLI that identified 514 differentially expressed genes (DEG). Gene ontology and pathway analysis of the DEG revealed transcripts among categories related to cell cycle, ribosome biogenesis, response to stress, cell migration, and cell proliferation. We further intersected the DEG in C2C12 myoblasts after LLLI with publicly available transcriptomes data from myogenic differentiation studies (myoblasts vs myotube) to identify transcripts with potential effects on myogenesis. This analysis revealed 42 DEG between myoblasts and myotube that intersect with altered genes in myoblasts after LLLI. Next, we performed a hierarchical cluster analysis with this set of shared transcripts that showed that LLLI myoblasts have a myotube-like profile, clustering away from the myoblast profile. The myotube-like transcriptional profile of LLLI myoblasts was further confirmed globally considering all the transcripts detected in C2C12 myoblasts after LLLI, by bi-dimensional clustering with myotubes transcriptional profiles, and by the comparison with 154 gene sets derived from previous published in vitro omics data. In conclusion, we demonstrate for the first time that LLLI regulates a set of mRNAs that control myoblast proliferation and differentiation into myotubes. Importantly, this set of mRNAs revealed a myotube-like transcriptional profile in LLLI myoblasts and provide new insights to the understanding of the molecular mechanisms underlying the effects of LLLI on skeletal muscle cells.