Automatic and unbiased segmentation and quantification of myofibers in skeletal muscle.
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ABSTRACT:
SUBMITTER: Waisman A
PROVIDER: S-EPMC8175575 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Skeletal muscle is an adaptive tissue with the ability to regenerate in response to exercise training. Cross-sectional area (CSA) quantification, as a main parameter to assess muscle regeneration capability, is highly tedious and time-consuming, necessitating an accurate and automated approach to analysis. Although several excellent programs are available to automate analysis of muscle histology, they fail to efficiently and accurately measure CSA in regenerating myofibers in response to exercise training. Here, we have developed a novel fully-automated image segmentation method based on neutrosophic set algorithms to analyse whole skeletal muscle cross sections in exercise-induced regenerating myofibers, referred as MyoView, designed to obtain accurate fiber size and distribution measurements. MyoView provides relatively efficient, accurate, and reliable measurements for CSA quantification and detecting different myofibers, myonuclei and satellite cells in response to the post-exercise regenerating process. We showed that MyoView is comparable with manual quantification. We also showed that MyoView is more accurate and efficient to measure CSA in post-exercise regenerating myofibers as compared with Open-CSAM, MuscleJ, SMASH and MyoVision. Furthermore, we demonstrated that to obtain an accurate CSA quantification of exercise-induced regenerating myofibers, whole muscle cross-section analysis is an essential part, especially for the measurement of different fiber-types. We present MyoView as a new tool to quantify CSA, myonuclei and satellite cells in skeletal muscle from any experimental condition including exercise-induced regenerating myofibers.
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Project description:Skeletal muscle myofibers have differential protein expression resulting in functionally distinct slow- and fast-twitch types. While certain protein classes are well-characterized, the depth of all proteins involved in this process is unknown. We utilized the Human Protein Atlas (HPA) and the HPASubC tool to classify mosaic expression patterns of staining across 49,600 unique tissue microarray (TMA) images using a visual proteomic approach. We identified 2164 proteins with potential mosaic expression, of which 1605 were categorized as "likely" or "real." This list included both well-known fiber-type-specific and novel proteins. A comparison of the 1605 mosaic proteins with a mass spectrometry (MS)-derived proteomic dataset of single human muscle fibers led to the assignment of 111 proteins to fiber types. We additionally used a multiplexed immunohistochemistry approach, a multiplexed RNA-ISH approach, and STRING v11 to further assign or suggest fiber types of newly characterized mosaic proteins. This visual proteomic analysis of mature skeletal muscle myofibers greatly expands the known repertoire of twitch-type-specific proteins.
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Project description:The abundance, anatomical distribution, and vascularity of skeletal muscle make it a potentially important contributor to local cytokine production and systemic cytokine abundance during inflammatory events. An orchestrated balance between the production of pro- and anti-inflammatory mediators is necessary for proper immune function, yet the contribution of the body's largest organ system, comprised primarily of skeletal muscle myocytes that fuse to form myofibers, to this process is largely unknown. Endotoxin (lipopolysaccharide, LPS) stimulates toll-like receptor 4 (TLR4) to induce the production of several pro-inflammatory cytokines, including interleukin-6 (IL-6) and C-C motif chemokine ligand 2 (CCL2), by a of myriad cell types. We sought to quantify the influence of myofibers on systemic cytokine concentrations following an acute endotoxemia challenge. To accomplish this, we generated muscle specific conditional knockouts for TLR4 (TLR4SMKO), IL-6 (IL6SMKO), and CCL2 (CCL2SMKO). We administered low concentrations of intravenous LPS (IV LPS) to these receptor and effector knockout mice and collected samples after 3 h. Using gene expression analysis of gastrocnemius muscle and serum cytokine measurements after IV LPS, we determined that deletion of myofiber IL-6 or CCL2 led to a 93% and 57% reduction of these specific cytokines in the systemic circulation, respectively. Myofiber specific TLR4 deletion decreased the expression of IL-6, CCL2, and C-X-C motif chemokine ligand 1 (CXCL1) in the gastrocnemius muscle. These data indicate the critical involvement and direct contribution of myofibers during the early systemic inflammatory cytokine response to endotoxin.
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Project description:BackgroundMaintaining stem cells in physiologically relevant states is necessary to understand cell and context-specific signalling paradigms and to understand complex interfaces between cells in situ. Understanding human stem cell function is largely based on tissue biopsies, cell culture, and transplantation into model organisms.MethodsHere, we describe a method to isolate post-mortem intact human muscle myofibers and culture muscle stem cells within the niche microenvironment to assay cellular dynamics, stem cell identity, stem cell hierarchy, and differentiation potential.ResultsWe show human myofiber culture maintains complex cell-cell contacts and extracellular niche composition during culture. Human satellite cells can be cultured at least 8 days, which represents a timepoint of activation, differentiation, and de novo human myofiber formation. We demonstrate that adult human muscle stem cells undergo apicobasal and planar cell divisions and express polarized dystrophin and EGFR. Furthermore, we validate that stimulation of the EGFR pathway stimulates the generation of myogenic progenitors and myogenic differentiation.ConclusionsThis method provides proof of principle evidence for the use of human muscle to evaluate satellite cell dynamics and has applications in pre-clinical evaluation of therapeutics targeting muscle repair.
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Project description:BackgroundSkeletal muscle is a complex, versatile tissue composed of a variety of functionally diverse fiber types. Although the biochemical, structural and functional properties of myofibers have been the subject of intense investigation for the last decades, understanding molecular processes regulating fiber type diversity is still complicated by the heterogeneity of cell types present in the whole muscle organ.Methodology/principal findingsWe have produced a first catalogue of genes expressed in mouse slow-oxidative (type 1) and fast-glycolytic (type 2B) fibers through transcriptome analysis at the single fiber level (microgenomics). Individual fibers were obtained from murine soleus and EDL muscles and initially classified by myosin heavy chain isoform content. Gene expression profiling on high density DNA oligonucleotide microarrays showed that both qualitative and quantitative improvements were achieved, compared to results with standard muscle homogenate. First, myofiber profiles were virtually free from non-muscle transcriptional activity. Second, thousands of muscle-specific genes were identified, leading to a better definition of gene signatures in the two fiber types as well as the detection of metabolic and signaling pathways that are differentially activated in specific fiber types. Several regulatory proteins showed preferential expression in slow myofibers. Discriminant analysis revealed novel genes that could be useful for fiber type functional classification.Conclusions/significanceAs gene expression analyses at the single fiber level significantly increased the resolution power, this innovative approach would allow a better understanding of the adaptive transcriptomic transitions occurring in myofibers under physiological and pathological conditions.
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