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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).
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 (<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.
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,
All procedures were approved by the Committee for Ethics in Experiments with animals of the University of Antioquia (UdeA) (minutes 104 of June 21st, 2016, and 005 of April 15th, 2021), according to Law 84 of 1989 and Resolution 8430 of 1993 issued by the Colombian Government and were performed and reported in compliance with the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines41. All results presented here come from healthy, 7-13 weeks old, 20-26 g.......
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 w.......
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 authors express their gratitude to Professor Robinson Ramírez 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), Medellín, Colombia, to JCC. Funders did not participate in data collection and analysis, manuscript writing or submission.
....Name | Company | Catalog Number | Comments |
Reagents | |||
Absolute ethanol | Sigma Aldrich | 32221 | |
Acetone | Merck | 179124 | |
Acrylamide | Gibco BRL | 15512-015 | |
Ammonium persulfate | Panreac | 141138.1610 | |
Anti myosin I antibody | Sigma Aldrich | M4276 | Primary antibody |
Anti myosin II antibody | Sigma Aldrich | M8421 | Primary antibody |
Anti myosin IIA antibody | American Type Culture Collection | SC-71 | Primary antibody. Derived from HB-277 hybridoma |
Anti myosin IIB antibody | Developmental Studies Hybridoma Bank | BF-F3-c | Primary antibody |
Bis-acrylamide | AMRESCO | 0172 | |
Bovine serum albumin | Thermo Scientific | B14 | |
Bradford reagent | Merck | 1.10306.0500 | |
Bromophenol blue | Carlo Erba | 428658 | |
Calcium carbonate | Merck | 102066 | |
Calcium dichloride (CaCl2) | Merck | 2389 | |
Chloroform | Sigma Aldrich | 319988 | |
Collagenase type 2 | Worthington | CLS-2/LS004176 | |
Consul-Mount | Thermo Scientific | 9990440 | |
Coomassie Brilliant blue R 250 | Merck | 112553 | |
Dimethyl sulfoxide (DMSO) | Sigma Aldrich | D2650 | |
Dithiothreitol (DTT) | AMRESCO | 0281 | |
Edetic acid (EDTA | AMRESCO | 0322 | |
Eosin Y | Sigma Aldrich | E4009 | |
Glycerol | Panreac | 1423291211 | |
Glycine | Panreac | 151340.1067 | |
Goat serum | Sigma Aldrich | G9023 | |
Hematoxylin | Thermo Scientific | 6765015 | |
HEPES | AMRESCO | 0511 | |
Hoechst 33258 | Sigma Aldrich | 861405 | |
Imidazole | AMRESCO | M136 | |
Isopentane | Sigma Aldrich | M32631 | |
Laminin | Sigma Aldrich | L2020 | |
Mag-Fluo-4, AM | Invitrogen | M14206 | Prepared only in DMSO. Pluronic acid is not required and should not be used to avoid fiber deterioration. |
Mercaptoethanol | Applichem | A11080100 | |
Methanol | Protokimica | MP10043 | |
Mice | Several | Several | 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. |
Mowiol 4-88 | Sigma Aldrich | 81381 | |
N,N,N',N'-tetramethylethane-1,2-diamine (TEMED) | Promega | V3161 | |
N-benzyl-p-toluene sulphonamide (BTS) | Tocris | 1870 | |
Optimal cutting compound (OCT) | Thermo Scientific | 6769006 | |
Secondary antibody | Thermo Scientific | A-11001 | Goat anti-mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 |
Sodium dodecil sulfate | Panreac | 1323631209 | |
TRIS 0.5 M, pH 6.8 | AMRESCO | J832 | |
Tris(Hydroxymethyl)aminomethane | AMRESCO | M151 | |
Triton X-100 | AMRESCO | M143 | |
Materials | |||
Dissection chamber | Custom-made | ||
Charged slides | Erie Scientific | 5951PLUS | |
Experimental bath chamber | Warner Instruments | RC-27NE2 | Narrow Bath Chamber with Field Stimulation, ensembled on a heated platform PH-6 |
Fine forceps | World Precision Instruments | 500338, 500230 | |
Fine scissors | World Precision Instruments | Vannas Scissors 501778 | |
Glass Pasteur pipettes | Several | Fire-polished tips | |
Glass vials with cap | Several | 2-3 mL volumen | |
Operating scissors | World Precision Instruments | 501223-G | |
Equipment | |||
Centrifuge | Thermo Scientific | SL 8R | |
Confocal microscope | Olympus | FV1000 | |
Cryostat | Leica | CM1850 | |
Digital camera | Zeiss | Erc 5s and Axio 305 | 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 |
Digitizer | Molecular Devices | 1550A Digidata | |
Electrophoresis chamber | Bio Rad | Mini-Protean IV | |
Inverted microscope coupled to fluorescence | Zeiss | Axio Observer A1 | Coupled to an appropriate light source, filters and objectives for fluorescence |
Photomultiplier | Horiba | R928 tube, Hamamatsu, in a D104 photometer, Horiba | Coupled to the lateral port of the fluorescence microscope |
Stereoscope | Zeiss | Stemi 508 | |
Stimulator | Grass Instruments | S6 | |
Water bath | Memmert | WNE-22 | |
Xilol | Sigma Aldrich | 808691 | |
Software | |||
Free software for electrophoreses analyses | University of Kentucky | GelBandFitter v1.7 | http://www.gelbandfitter.org |
Free software for image analysis and morphometry | National Institutes of Health | ImageJ v1.54 | https://imagej.nih.gov/ij/index.html |
Licensed software for Ca2+ signals acquisition and analyses | Molecular Devices | pCLAMP v10.05 | https://www.moleculardevices.com |
Licensed software for statistical analyses and graphing | OriginLab | OriginPro 2019 | https://www.originlab.com/ |
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