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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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).

Abstract

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.

Introduction

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,

Protocol

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.......

Representative Results

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.......

Discussion

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 .......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
Reagents
Absolute ethanolSigma Aldrich32221
AcetoneMerck179124
AcrylamideGibco BRL15512-015
Ammonium persulfatePanreac141138.1610
Anti myosin I antibodySigma AldrichM4276Primary antibody
Anti myosin II antibodySigma AldrichM8421Primary antibody
Anti myosin IIA antibodyAmerican Type Culture CollectionSC-71Primary antibody. Derived from HB-277 hybridoma
Anti myosin IIB antibodyDevelopmental Studies Hybridoma BankBF-F3-c Primary antibody
Bis-acrylamideAMRESCO0172
Bovine serum albuminThermo ScientificB14
Bradford reagentMerck1.10306.0500
Bromophenol blueCarlo Erba428658
Calcium carbonateMerck102066
Calcium dichloride (CaCl2)Merck2389
ChloroformSigma Aldrich319988
Collagenase type 2WorthingtonCLS-2/LS004176
Consul-MountThermo Scientific9990440
Coomassie Brilliant blue R 250 Merck112553
Dimethyl sulfoxide (DMSO)Sigma AldrichD2650
Dithiothreitol (DTT)AMRESCO0281
Edetic acid (EDTAAMRESCO0322
Eosin YSigma AldrichE4009
GlycerolPanreac 1423291211
GlycinePanreac151340.1067
Goat serumSigma AldrichG9023
HematoxylinThermo Scientific6765015
HEPESAMRESCO0511
Hoechst 33258Sigma Aldrich861405
ImidazoleAMRESCOM136
IsopentaneSigma AldrichM32631
LamininSigma AldrichL2020
Mag-Fluo-4, AMInvitrogenM14206Prepared only in DMSO. Pluronic acid is not required and should not be used to avoid fiber deterioration.
MercaptoethanolApplichemA11080100
MethanolProtokimicaMP10043
MiceSeveralSeveralFor 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 4-88Sigma Aldrich81381
N,N,N',N'-tetramethylethane-1,2-diamine (TEMED)PromegaV3161
N-benzyl-p-toluene sulphonamide (BTS)Tocris1870
Optimal cutting compound (OCT)Thermo Scientific6769006
Secondary antibodyThermo ScientificA-11001Goat anti-mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488
Sodium dodecil sulfatePanreac 1323631209
TRIS 0.5 M, pH 6.8 AMRESCOJ832
Tris(Hydroxymethyl)aminomethaneAMRESCOM151
Triton X-100AMRESCOM143
Materials
Dissection chamberCustom-made
Charged slidesErie Scientific5951PLUS
Experimental bath chamberWarner InstrumentsRC-27NE2Narrow Bath Chamber with Field Stimulation, ensembled on a heated platform PH-6
Fine forcepsWorld Precision Instruments500338, 500230
Fine scissorsWorld Precision InstrumentsVannas Scissors 501778
Glass Pasteur pipettesSeveralFire-polished tips
Glass vials with capSeveral2-3 mL volumen
Operating scissorsWorld Precision Instruments501223-G
Equipment
CentrifugeThermo ScientificSL 8R
Confocal microscopeOlympusFV1000
CryostatLeicaCM1850
Digital cameraZeissErc 5s and Axio 305Axio 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
DigitizerMolecular Devices1550A Digidata
Electrophoresis chamberBio RadMini-Protean IV
Inverted microscope coupled to fluorescenceZeissAxio Observer A1Coupled to an appropriate light source, filters and objectives for fluorescence
PhotomultiplierHoribaR928 tube, Hamamatsu, in a D104 photometer, HoribaCoupled to the lateral port of the fluorescence microscope
StereoscopeZeissStemi 508
Stimulator Grass Instruments S6
Water bath MemmertWNE-22
XilolSigma Aldrich808691
Software
Free software for electrophoreses analysesUniversity of KentuckyGelBandFitter v1.7http://www.gelbandfitter.org
Free software for image analysis and morphometryNational Institutes of HealthImageJ v1.54https://imagej.nih.gov/ij/index.html
Licensed software for Ca2+ signals acquisition and analysesMolecular DevicespCLAMP v10.05https://www.moleculardevices.com
Licensed software for statistical analyses and graphingOriginLabOriginPro 2019https://www.originlab.com/

References

  1. Frontera, W. R., Ochala, J. Skeletal muscle: a brief review of structure and function. Calcif Tissue Int. 96 (3), 183-195 (2015).
  2. Barclay, C., Launikonis, B. Components of activation heat in skeletal muscle. J M....

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