The authors express their gratitude to Professor Robinson Ram&#237;rez from UdeA for help with animals and some photos and to Carolina Palacios for technical support. Johan Pineda from Kaika helped us to set up the color and fluorescence cameras. Shyuan Ngo, from the University of Queensland, kindly proofread the manuscript. This study was funded by the CODI-UdeA (2020-34909 from February 22nd, 2021, and 2021-40170 from March 31st, 2022, SIU), and Planning Office-UdeA (E01708-K and ES03180101), Medell&#237;n, Colombia, to JCC. Funders did not participate in data collection and analysis, manuscript writing or submission.

Enzymatic Isolation and Characterization of Variable Length Mouse Muscle Fibers

The authors declare that they have no conflicts of interest.

To complement the models available for studying mature skeletal muscle biology, here we demonstrate the successful enzymatic dissociation of a range of mouse muscles with short, intermediate, and long fibers. These fibers allow for the demonstration of the generalizability of the MT-II kinetics of the Ca2+ transients in skeletal muscle. Further, the fiber types in the intact, whole muscles were classified. Given that the FDB is the most used muscle for physiological experiments, the types of fibers present in ...

The mature skeletal muscle of mammals is a multifunctional tissue. It heavily regulates metabolism, is the main source of heat production, and its dynamical properties confer upon it a key role in respiration, movement of body segments, or displacement from one point to another1,2,3. Skeletal muscle is also relevant for the pathophysiology of many illnesses, including inherited and chronic conditions, such as myopathies, dystrophies, or sarcopenia, as well as many non-muscle chronic conditions, such as cardiometabolic diseases3,4,5,6,7,8.
The ex vivo study of the structural and functional properties of mature skeletal muscle in the context of health and disease has been possible mainly through two experimental models: whole muscle and isolated fibers. In the 20th century, researchers exploited the properties of the whole, intact extensor digitorum longus (EDL), soleus, tibialis anterior, and gastrocnemius muscles of different small species as pivotal models to learn about motor units, fiber types, and dynamic properties such as force and kinetics of contraction and relaxation9,10,11,12,13,14,15,16. However, the advent of more refined cell biology studies moved the area toward the study of single muscle fibers. Pioneering work then enabled the isolation of intact flexor digitorum brevis (FDB) fibers of rats by enzymatic dissociation for subsequent characterization17,18,19. Although FDB fibers can also be obtained by manual dissection20, the ease and high throughput of enzymatic dissociation of murine muscles, in addition to their suitability for a variety of experimental approaches, have made the latter model widely used during the last two decades.
The short FDB fibers are suitable for electrophysiological and other biophysical studies, biochemical, metabolic, and pharmacological analyses, electron and fluorescence microscopy experiments, transfection for cell biology approaches, or as a source of stem cells in myogenesis studies5,21,22,23,24,25,26,27,28,29,30,31,32. However, using only FDB fibers in muscle experiments narrows the scope of research dealing with fiber types and limits the amount of biological material available for some methodological techniques or for gaining more information from one animal. These limitations hinder a clear correlation of cellular physiological phenomena with previous biochemical and dynamical studies performed in different whole, intact, muscles (e.g., EDL, soleus, peronei).
Overcoming these limitations, some groups succeeded in dissociating the longer EDL and soleus muscles24,33,34,35,36,37,38,39,40, opening the door to further extend the method to other relevant muscles. However, the use of EDL and soleus fibers is still scarce, likely due to the lack of methodological details for getting them as intact fibers. Here, we describe in detail how to isolate fibers of different lengths and types from six muscles: three of them already described (FDB, EDL, and soleus) and three of them successfully dissociated for the first time (extensor hallucis longus [EHL], peroneus longus [PL], and peroneus digiti quarti [PDQA]). The results of the present work confirm that the model of enzymatically dissociated fibers is apt for a wide range of studies and future correlations with previously published data, thus increasing the availability of models for mature skeletal muscle studies.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Absolute ethanol</td><td>Sigma Aldrich</td><td>32221</td><td></td></tr><tr><td>Acetone</td><td>Merck</td><td>179124</td><td></td></tr><tr><td>Acrylamide</td><td>Gibco BRL</td><td>15512-015</td><td></td></tr><tr><td>Ammonium persulfate</td><td>Panreac</td><td>141138.1610</td><td></td></tr><tr><td>Anti myosin I antibody</td><td>Sigma Aldrich</td><td>M4276</td><td>Primary antibody</td></tr><tr><td>Anti myosin II antibody</td><td>Sigma Aldrich</td><td>M8421</td><td>Primary antibody</td></tr><tr><td>Anti myosin IIA antibody</td><td>American Type Culture Collection</td><td>SC-71</td><td>Primary antibody. Derived from HB-277 hybridoma</td></tr><tr><td>Anti myosin IIB antibody</td><td>Developmental Studies Hybridoma Bank</td><td>BF-F3-c&amp;nbsp;</td><td>Primary antibody</td></tr><tr><td>Bis-acrylamide</td><td>AMRESCO</td><td>0172</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Thermo Scientific</td><td>B14</td><td></td></tr><tr><td>Bradford reagent</td><td>Merck</td><td>1.10306.0500</td><td></td></tr><tr><td>Bromophenol blue</td><td>Carlo Erba</td><td>428658</td><td></td></tr><tr><td>Calcium carbonate</td><td>Merck</td><td>102066</td><td></td></tr><tr><td>Calcium dichloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Merck</td><td>2389</td><td></td></tr><tr><td>Chloroform</td><td>Sigma Aldrich</td><td>319988</td><td></td></tr><tr><td>Collagenase type 2</td><td>Worthington</td><td>CLS-2/LS004176</td><td></td></tr><tr><td>Consul-Mount</td><td>Thermo Scientific</td><td>9990440</td><td></td></tr><tr><td>Coomassie Brilliant blue R 250&amp;nbsp;</td><td>Merck</td><td>112553</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma Aldrich</td><td>D2650</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>AMRESCO</td><td>0281</td><td></td></tr><tr><td>Edetic acid (EDTA</td><td>AMRESCO</td><td>0322</td><td></td></tr><tr><td>Eosin Y</td><td>Sigma Aldrich</td><td>E4009</td><td></td></tr><tr><td>Glycerol</td><td>Panreac&amp;nbsp;</td><td>1423291211</td><td></td></tr><tr><td>Glycine</td><td>Panreac</td><td>151340.1067</td><td></td></tr><tr><td>Goat serum</td><td>Sigma Aldrich</td><td>G9023</td><td></td></tr><tr><td>Hematoxylin</td><td>Thermo Scientific</td><td>6765015</td><td></td></tr><tr><td>HEPES</td><td>AMRESCO</td><td>0511</td><td></td></tr><tr><td>Hoechst 33258</td><td>Sigma Aldrich</td><td>861405</td><td></td></tr><tr><td>Imidazole</td><td>AMRESCO</td><td>M136</td><td></td></tr><tr><td>Isopentane</td><td>Sigma Aldrich</td><td>M32631</td><td></td></tr><tr><td>Laminin</td><td>Sigma Aldrich</td><td>L2020</td><td></td></tr><tr><td>Mag-Fluo-4, AM</td><td>Invitrogen</td><td>M14206</td><td>Prepared only in DMSO. Pluronic acid is not required and should not be used to avoid fiber deterioration.</td></tr><tr><td>Mercaptoethanol</td><td>Applichem</td><td>A11080100</td><td></td></tr><tr><td>Methanol</td><td>Protokimica</td><td>MP10043</td><td></td></tr><tr><td>Mice</td><td>Several</td><td>Several</td><td>For this manuscript, we only used C57BL/6 mice. However, some preliminary results have shown that the protocol works well for Swiss Webster mice of the same age and weight.</td></tr><tr><td>Mowiol 4-88</td><td>Sigma Aldrich</td><td>81381</td><td></td></tr><tr><td>N,N,N&#039;,N&#039;-tetramethylethane-1,2-diamine (TEMED)</td><td>Promega</td><td>V3161</td><td></td></tr><tr><td>N-benzyl-p-toluene sulphonamide (BTS)</td><td>Tocris</td><td>1870</td><td></td></tr><tr><td>Optimal cutting compound (OCT)</td><td>Thermo Scientific</td><td>6769006</td><td></td></tr><tr><td>Secondary antibody</td><td>Thermo Scientific</td><td>A-11001</td><td>Goat anti-mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td></tr><tr><td>Sodium dodecil sulfate</td><td>Panreac&amp;nbsp;</td><td>1323631209</td><td></td></tr><tr><td>TRIS 0.5 M, pH 6.8&amp;nbsp;</td><td>AMRESCO</td><td>J832</td><td></td></tr><tr><td>Tris(Hydroxymethyl)aminomethane</td><td>AMRESCO</td><td>M151</td><td></td></tr><tr><td>Triton X-100</td><td>AMRESCO</td><td>M143</td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dissection chamber</td><td>Custom-made</td><td></td><td></td></tr><tr><td>Charged slides</td><td>Erie Scientific</td><td>5951PLUS</td><td></td></tr><tr><td>Experimental bath chamber</td><td>Warner Instruments</td><td>RC-27NE2</td><td>Narrow Bath Chamber with Field Stimulation, ensembled on a heated platform PH-6</td></tr><tr><td>Fine forceps</td><td>World Precision Instruments</td><td>500338, 500230</td><td></td></tr><tr><td>Fine scissors</td><td>World Precision Instruments</td><td>Vannas Scissors 501778</td><td></td></tr><tr><td>Glass Pasteur pipettes</td><td>Several</td><td></td><td>Fire-polished tips</td></tr><tr><td>Glass vials with cap</td><td>Several</td><td></td><td>2-3 mL volumen</td></tr><tr><td>Operating scissors</td><td>World Precision Instruments</td><td>501223-G</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Thermo Scientific</td><td>SL 8R</td><td></td></tr><tr><td>Confocal microscope</td><td>Olympus</td><td>FV1000</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM1850</td><td></td></tr><tr><td>Digital camera</td><td>Zeiss</td><td>Erc 5s and Axio 305</td><td>Axio 305, coupled to the Stemi 508 stereoscope, was used to take pictures during dissection; while Erc 5s or Axio 208, coupled to the Axio Observer A1 microscope, were used to take images of the isolated fibers and the immunofluorescence assays</td></tr><tr><td>Digitizer</td><td>Molecular Devices</td><td>1550A Digidata</td><td></td></tr><tr><td>Electrophoresis chamber</td><td>Bio Rad</td><td>Mini-Protean IV</td><td></td></tr><tr><td>Inverted microscope coupled to fluorescence</td><td>Zeiss</td><td>Axio Observer A1</td><td>Coupled to an appropriate light source, filters and objectives for fluorescence</td></tr><tr><td>Photomultiplier</td><td>Horiba</td><td>R928 tube, Hamamatsu, in a D104 photometer, Horiba</td><td>Coupled to the lateral port of the fluorescence microscope</td></tr><tr><td>Stereoscope</td><td>Zeiss</td><td>Stemi 508</td><td></td></tr><tr><td>Stimulator&amp;nbsp;</td><td>Grass Instruments&amp;nbsp;</td><td>S6</td><td></td></tr><tr><td>Water bath&amp;nbsp;</td><td>Memmert</td><td>WNE-22</td><td></td></tr><tr><td>Xilol</td><td>Sigma Aldrich</td><td>808691</td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Free software for electrophoreses analyses</td><td>University of Kentucky</td><td>GelBandFitter v1.7</td><td>http://www.gelbandfitter.org</td></tr><tr><td>Free software for image analysis and morphometry</td><td>National Institutes of Health</td><td>ImageJ v1.54</td><td>https://imagej.nih.gov/ij/index.html</td></tr><tr><td>Licensed software for Ca&lt;sup&gt;2+&lt;/sup&gt; signals acquisition and analyses</td><td>Molecular Devices</td><td>pCLAMP v10.05</td><td>https://www.moleculardevices.com</td></tr><tr><td>Licensed software for statistical analyses and graphing</td><td>OriginLab</td><td>OriginPro 2019</td><td>https://www.originlab.com/</td></tr></tbody></table>

intact short, intermediate, and long skeletal muscle fibers obtained by enzymatic dissociation of six hindlimb muscles of mice: beyond flexor digitorum brevis

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal with the short fibers of the flexor digitorum brevis (FDB), which restrains the scope of results dealing with fiber types, limits the amount of biological material available, and impedes a clear connection between cellular physiological phenomena and previous biochemical and dynamical knowledge obtained in other muscles.
This paper describes how to obtain intact fibers from six muscles with different fiber type profiles and lengths. Using C57BL/6 adult mice, we show the muscle dissection and fiber isolation protocol and demonstrate the suitability of the fibers for Ca2+ transient studies and their morphometric characterization. The fiber type composition of the muscles is also presented. When dissociated, all muscles rendered intact, living fibers that contract briskly for more than 24 h. FDB gave short (&#60;1 mm), peroneus digiti quarti (PDQA) and peroneus longus (PL) gave intermediate (1-3 mm), while extensor digitorum longus (EDL), extensor hallucis longus (EHL), and soleus muscles released long (3-6 mm) fibers.
When recorded with the fast dye Mag-Fluo-4, Ca2+ transients of PDQA, PL, and EHL fibers showed the fast, narrow kinetics reminiscent of the morphology type II (MT-II), known to correspond to type IIX and IIB fibers. This is consistent with the fact that these muscles have over 90% of type II fibers compared with FDB (~80%) and soleus (~65%). Moving beyond FDB, we demonstrate for the first time the dissociation of several muscles, which render fibers spanning a range of lengths between 1 and 6 mm. These fibers are viable and give fast Ca2+ transients, indicating that the MT-II can be generalized to IIX and IIB fast fibers, regardless of their muscle source. These results increase the availability of models for mature skeletal muscle studies.

Sarcoplasmic Ca2+ concentration during a twitch 
To demonstrate the feasibility of physiological experiments in the set of dissociated fibers and to extend our previous findings on excitation-contraction coupling (ECC) and fiber types, Ca2+ transients were acquired in fibers from all muscles. First, FDB (n = 5) and EDL (n = 7) showed Ca2+ kinetics known as morphology type II (MT-II). These are fast, spiky signals, whose RT lasts ~1 ms; its decay phase can be fitted wit...

Watch this Scientific Journal Video about Intact Short, Intermediate, and Long Skeletal Muscle Fibers Obtained by Enzymatic Dissociation of Six Hindlimb Muscles of Mice: Beyond Flexor Digitorum Brevis

Author Spotlight: Isolation of Long Muscle Fibers from Mouse Hindlimb Muscles for Studying Excitation-Contraction Coupling Across Fiber Types

We describe a protocol to obtain enzymatically dissociated fibers of different lengths and types from six muscles of adult mice: three of them already described (flexor digitorum brevis, extensor digitorum longus, soleus) and three of them successfully dissociated for the first time (extensor hallucis longus, peroneus longus, peroneus digiti quarti).

Intact Short, Intermediate, and Long Skeletal Muscle Fibers Obtained by Enzymatic Dissociation of Six Hindlimb Muscles of Mice: Beyond Flexor Digitorum Brevis

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal ...

intact-short-intermediate-long-skeletal-muscle-fibers-obtained

Research

JoVE Journal

Biology

1.7K Views.  University of Antioquia. In deze studie beschrijven we een methode om vezels van verschillende lengtes en typen te isoleren uit zes achterpootspieren van muizen. Ook testten we de geschiktheid van de vezels voor fysiologische experimenten die te maken hadden met de studie van het excitatie-contractiekoppelingsmechanisme. Jarenlang was het verkrijgen van lange spiervezels een uitdaging, waardoor de reikwijdte van veel onderzoeken in het veld werd beperkt.Nu laten we zien dat het mogelijk is om magere vezels te ...

Video: Auteur Spotlight: Isolatie van lange spiervezels uit de achterpoten van muizen voor het bestuderen van excitatie-contractiekoppeling over vezeltypen

X-ray Diffraction of Intact Murine Skeletal Muscle as a Tool for Studying the Structural Basis of Muscle Disease

Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases including those of skeletal muscle. Mouse skeletal muscle has been shown to produce high quality X-ray diffraction patterns on third generation synchrotron beamlines providing an opportunity to link changes at the level of the genotype to functional phenotypes in health and disease by determining the structural consequences of genetic changes. We present detailed protocols for preparation of specimens, collecting the X-ray patterns and extracting relevant structural parameters from the X-ray patterns that may help guide experimenters wishing to perform such experiments for themselves.

We present detailed protocols for performing small-angle X-ray diffraction experiments using intact mouse skeletal muscles. With the wide availability of transgenic mouse models for human diseases, this experimental platform can form a useful test bed for elucidating the structural basis of genetic muscle diseases

Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases including those of skeletal ...

Developmental Biology

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

Critical to the evaluation of potential therapeutics for muscular disease are sensitive and reproducible physiological assessments of muscle function. Because many pre-clinical trials rely on mouse models for these diseases, isolated muscle function has become one of the standards for Go/NoGo decisions in moving drug candidates forward into patients. We will demonstrate the preparation of the extensor digitorum longus (EDL) and diaphragm muscles for functional testing, which are the predominant muscles utilized for these studies. The EDL muscle geometry is ideal for isolated muscle preparations, with two easily accessible tendons, and a small size that can be supported by superfusion in a bath. The diaphragm exhibits profound progressive pathology in dystrophic animals, and can serve as a platform for evaluating many potential therapies countering fibrosis, and promoting myofiber stability. Protocols for routine testing, including isometric and eccentric contractions, will be shown. Isometric force provides assessment of strength, and eccentric contractions help to evaluate sarcolemma stability, which is disrupted in many types of muscular dystrophies. Comparisons of the expected results between muscles from wildtype and dystrophic muscles will also be provided. These measures can complement morphological and biochemical measurements of tissue homeostasis, as well as whole animal assessments of muscle function.

Muscle function measurements contribute to the evaluation of potential therapeutics for muscle pathology, as well as to the determination of mechanisms underlying physiology of this tissue. We will demonstrate the preparation of the extensor digitorum longus and diaphragm muscles for functional testing. Protocols for isometric and eccentric contractions will be shown, as well as differences in results between dystrophic muscles, representing a pathological state, and wildtype muscles.

Critical to the evaluation of potential  therapeutics for muscular disease are sensitive and reproducible physiological  assessments of muscle function. ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

Neuroscience

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

Medicine

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...

Functional Site-Directed Fluorometry in Native Skeletal Muscle Cells

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability

Functional site-directed fluorometry has been the technique of choice to investigate the structure-function relationship of numerous membrane proteins, including voltage-gated ion channels. This approach has been used primarily in heterologous expression systems to simultaneously measure membrane currents, the electrical manifestation of the channels&#39; activity, and fluorescence measurements, reporting local domain rearrangements. Functional site-directed fluorometry combines electrophysiology, molecular biology, chemistry, and fluorescence into a single wide-ranging technique that permits the study of real-time structural rearrangements and function through fluorescence and electrophysiology, respectively. Typically, this approach requires an engineered voltage-gated membrane channel that contains a cysteine that can be tested by a thiol-reactive fluorescent dye. Until recently, the thiol-reactive chemistry used for the site-directed fluorescent labeling of proteins was carried out exclusively in Xenopus oocytes and cell lines, restricting the scope of the approach to primary non-excitable cells. This report describes the applicability of functional site-directed fluorometry in adult skeletal muscle cells to study the early steps of excitation-contraction coupling, the process by which muscle fiber electrical depolarization is linked to the activation of muscle contraction. The present protocol describes the methodologies to design and transfect cysteine-engineered voltage-gated Ca2+ channels (CaV1.1) into muscle fibers of the flexor digitorum brevis of adult mice using in vivo electroporation and the subsequent steps required for functional site-directed fluorometry measurements. This approach can be adapted to study other ion channels and proteins. The use of functional site-directed fluorometry of mammalian muscle is particularly relevant to studying basic mechanisms of excitability.

Author Spotlight: Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability  

Functional site-directed fluorometry is a method to study protein domain motions in real time. Modification of this technique for its application in native cells now allows the detection and tracking of single voltage-sensor motions from voltage-gated Ca2+ channels in murine isolated skeletal muscle fibers.

Functional site-directed fluorometry has been the technique of choice to investigate the structure-function relationship of numerous membrane proteins, ...

Optimized Isolation of Skeletal Muscle-Derived EVs for Research and Therapeutics

Enzymatic Isolation of Skeletal Muscle Interstitial Extracellular Vesicles

Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released by cells to transport bioactive cargo, such as proteins, RNAs, and DNAs, for intercellular communication. Investigating EV-mediated crosstalk among cells in muscle homeostasis and diseases offers significant potential to enhance our understanding of muscle development, regeneration, and atrophy. However, current protocols for isolating skeletal muscle-derived EVs (SkM-EVs) face challenges in achieving high purity and yield, primarily due to difficulties in releasing EVs from muscle tissues without compromising cellular membranes. This article presents an efficient protocol for SkM-EV isolation, comprising mechanical detachment, enzymatic dissociation, filtration, and ultracentrifugation. These steps are optimized to enhance EV release from muscle tissues, yielding high-purity SkM-EVs. Subsequently, nano-flow cytometry, BCA assay, and Western blot assay are performed to characterize the quantity and quality of the isolated SkM-EVs. This protocol holds promise for establishing a reliable platform to obtain tissue-derived EVs for advancing basic research, disease diagnosis, and drug delivery.

This protocol aims to isolate and purify skeletal muscle interstitial extracellular vesicles (SkM-EVs) from rodent muscle tissues through mechanical detachment, enzymatic dissociation, filtration, and differential ultracentrifugation. It demonstrates consistency and reliability. The isolated SkM-EVs provide insights into muscle homeostasis and diseases, with potential applications as diagnostic biomarkers or therapeutic vehicles.

Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released by cells to transport bioactive cargo, such as proteins, RNAs, and DNAs, ...

Hindlimb Skeletal Muscle Extraction: A Procedure to Excise Hindlimb Skeletal Muscles from Murine Model

Source: Bonetto, A. et al. <a href="https://www.jove.com/video/54893" target="_blank">The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia.</a> J. Vis. Exp. (2016)
In this video, we describe the procedure to excise hindlimb skeletal muscles from a mouse model. The excised muscles can be stored at lower temperatures until further downstream analysis.&#160;

Source: Bonetto, A. et al. The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia. J. Vis. Exp. (2016)
In this video, we ...

Intact Short, Intermediate, and Long Skeletal Muscle Fibers Obtained by Enzymatic Dissociation of Six Hindlimb Muscles of Mice: Beyond Flexor Digitorum Brevis

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