Automatic and unbiased segmentation and quantification of myofibers in skeletal muscle

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.Fil: Waisman, Ariel. Fundacion P/la Lucha C/enferm.neurologicas Infancia. Instituto de Neurociencias. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Instituto de Neurociencias.; ArgentinaFil: Norris, Alessandra. University of Florida; Estados UnidosFil: Elias Costa, Martin. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Kopinke, Daniel. University of Florida; Estados Unido

Kehalise aktiivsuse preventiivne mõju sarkopeeniaga kaasnevatele muutustele

http://tartu.ester.ee/record=b2655494~S1*es

β-CATENIN REGULATION OF ADULT SKELETAL MUSCLE PLASTICITY

Adult skeletal muscle is highly plastic and responds readily to environmental stimuli. One of the most commonly utilized methods to study skeletal muscle adaptations is immunofluorescence microscopy. By analyzing images of adult muscle cells, also known as myofibers, one can quantify changes in skeletal muscle structure and function (e.g. hypertrophy and fiber type). Skeletal muscle samples are typically cut in transverse or cross sections, and antibodies against sarcolemmal or basal lamina proteins are used to label the myofiber boundaries. The quantification of hundreds to thousands of myofibers per sample is accomplished either manually or semi-automatically using generalized pathology software, and such approaches become exceedingly tedious. In the first study, I developed MyoVision, a robust, fully automated software that is dedicated to skeletal muscle immunohistological image analysis. The software has been made freely available to muscle biologists to alleviate the burden of routine image analyses. To date, more than 60 technicians, students, postdoctoral fellows, faculty members, and others have requested this software. Using MyoVision, I was able to accurately quantify the effects of β-catenin knockout on myofiber hypertrophy. In the second study, I tested the hypothesis that myofiber hypertrophy requires β-catenin to activate c-myc transcription and promote ribosome biogenesis. Recent evidence in both mice and human suggests a close association between ribosome biogenesis and skeletal muscle hypertrophy. Using an inducible mouse model of skeletal myofiber-specific genetic knockout, I obtained evidence that β-catenin is important for myofiber hypertrophy, although its role in ribosome biogenesis appears to be dispensable for mechanical overload induced muscle growth. Instead, β-catenin may be necessary for promoting the translation of growth related genes through activation of ribosomal protein S6. Unexpectedly, we detected a novel, enhancing effect of myofiber β-catenin knockout on the resident muscle stem cells, or satellite cells. In the absence of myofiber β-catenin, satellite cells activate and proliferate earlier in response to mechanical overload. Consistent with the role of satellite cells in muscle repair, the enhanced recruitment of satellite cells led to a significantly improved regeneration response after chemical injury. The novelty of these findings resides in the fact that the genetic perturbation was extrinsic to the satellite cells, and this is even more surprising because the current literature focuses heavily on intrinsic mechanisms within satellite cells. As such, this model of myofiber β-catenin knockout may significantly contribute to better understanding of the mechanisms of satellite cell priming, with implications for regenerative medicine

Myonuclear dynamics in muscle plasticity and the transcriptional regulation of resistance training induced hypertrophy.

Skeletal muscle is highly responsive to changes in mechanical load or activity and can adjust its morphological, metabolic, and contractile properties accordingly. The remodeling of these characteristics is controlled by the reprogramming of the transcriptional output of the myonuclei along the length of the muscle fiber. To meet the transcriptional demands of growth and increased activity, myonuclei can be added to the existing cytoplasm through the fusion of satellite cells, to support synthetic activity. This project utilises improved methodologies including automated, high-throughput immunohistochemical analysis and bulk RNA-sequencing of skeletal muscle. With these techniques, we define the temporal patterns of myonuclear dynamics and how they correspond to fiber-type specific adaptations in response to loading, unloading, reloading and changes in activity and how the acute transcriptional response is altered, dependent on the training status of the muscle. To induce these modalities of activity or inactivity, we utilised in-vivo models from our lab including, (1) programmed exercise delivered through miniature implanted pulse generators (IPGs) to induce muscle hypertrophy or metabolic adaptation and (2) disuse by means of tetrodotoxin-induced nerve silencing to induce muscle atrophy. We report that the genes that most closely track with changes in muscle mass are controlled centrally by the basic-helix-loop-helix transcription factor Myc, that functions to bind to E-box containing DNA sequences. In addition, we identify 10 other genes that appear as important regulators across species and modalities of exercise that warrant further investigation. Lastly, we investigate a promising marker for specifically identifying myonuclei, pericentriolar material-1 (PCM1), which would allow for deconvolution of mRNA signals from bulk skeletal muscle mRNA analysis, allowing for identification of myogenic and non-myogenic transcriptional changes. In summary, our aim is to provide key mechanistic insights into myonuclear dynamics and how adaptation of skeletal muscle is regulated at the transcriptional level