This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. This project was supported by grant P41 GM103622 from the National Institute of General Medical Sciences of the National Institutes of Health. Use of the Pilatus 3 1M detector was provided by grant 1S10OD018090-01 from NIGMS. The content is solely the responsibility of the authors and does not necessarily reflect the official views of the National Institute of General Medical Sciences or the National Institutes of Health.

All animal experiments protocols were approved by the Illinois Institute of Technology Institutional Animal Care and Use Committee (Protocol 2015-001, Approval date: 3 November 2015) and followed the NIH &#34;Guide for the Care and Use of Laboratory Animals&#34;9.
1. Pre-experiment Preparation
<ol>
	<li>Prepare 500 mL of Ringer&#8217;s solution (contains: 145 mM NaCl, 2.5 mM KCl, 1.0 mM MgSO4, 1.0 mM CaCl2, 10.0 mM HEPES, 11 mM glucose, pH 7.4) freshly for each day of the experiment.</li>
	<li>Fill 200 mL of Ringer&#39;s solution in a spray bottle and store at 4 &#176;C fridge. Fill a Petri dish (10 cm in diameter) with Ringer&#39;s solution and perfuse with 100% oxygen by connecting the tube from an oxygen cylinder to an aquarium air stone. The Petri dishes (&#34;dissecting dishes&#34;) were previously coated with an elastomer compound to allow inserting pins during the dissection.</li>
	<li>Prepare metal mounting hooks. Cut two pieces of stainless-steel wire, 0.5 mm in diameter, to the appropriate length and bend the wire at both ends to form hooks. Arrange all the dissecting tools, scissors, suture tying forceps, micro-scissors handy for use. 
	NOTE: The hook part should be about 3 mm long. The longer wire (ending in a hook) should be about&#160;5 cm&#160;long, and the shorter wire (also ending in a hook) should be about&#160;1 cm&#160;long in order to fit the custom chambers used at BioCAT and allow for a sufficient range of motion for the transducer arm.&#160;</li>
	<li>Connect and turn on all the equipment. This includes a combined motor/force transducer, motor/force transducer controller a high-power bi-phasic current stimulator, and a computer controlled data acquisition/control system.
	<ol>
		<li>Turn on the data acquisition system and calibrate it before beginning the experiment10. Briefly, calibrating the force by adding a set of known weights, covering up to 50% of the maximum force measured by the force transducer in a linear progression, on the force transducer and recording the output voltage changes. Calibrate the length by applying a set of known output voltage to the lever arm and measure the length change of the arm.</li>
		<li>Connect the hoses from the thermal block on the sample holder to a refrigerated circulating bath and set the temperature to maintain the desired temperature in the chamber to between 10 &#176;C and 40 &#176;C. Determine this empirically ahead of time by setting the circulating bath to a range of temperatures and measuring the temperature in the chamber with a thermocouple.</li>
	</ol>
	</li>
</ol>
2. Muscle Preparation
<ol>
	<li>Euthanizing the mouse
	<ol>
		<li>Euthanize the mouse by carbon dioxide inhalation followed by cervical dislocation.</li>
		<li>Spray the skin on the hind limb with cold Ringer&#8217;s solution to prevent hair from blowing into the preparation. Remove the skin by cutting it away around the thigh using fine dissection scissors and quickly pull the skin down using #5 forceps to expose the muscles.</li>
		<li>Amputate the hind limb and transfer it to a dissecting dish that has been filled with oxygenized Ringer&#8217;s solution, and then place under a binocular dissecting microscope.</li>
	</ol>
	</li>
	<li>Preparing a soleus muscle
	<ol>
		<li>Pin the hind limb down in the dissecting dish with the gastrocnemius muscle facing upwards. Cut the distal tendon of the gastrocnemius/soleus muscle group and lift the muscles gently and slowly by cutting away the fascia on either side of the gastrocnemius muscle using fine scissors. Isolate the gastrocnemius/soleus muscle group from the limb after freeing the proximal tendon of the soleus muscle.</li>
		<li>Pin the muscle group containing the gastrocnemius muscle and the distal tendon down in the dissecting dish. Lift the soleus muscle gently via the proximal tendon and separate it from the gastrocnemius muscle leaving as much of the soleus distal tendon intact as possible.</li>
	</ol>
	</li>
	<li>Preparing an extensor digitorium longus (EDL) muscle
	<ol>
		<li>Pin the hind limb down in the dissecting dish with the tibialis anterior muscle facing upwards. Cut the fascia along the tibialis anterior (TA) muscle and pull it clear using forceps. Identify and cut the distal tendon of the TA muscle. Lift the TA muscle and cut it out carefully without pulling on the EDL muscle.</li>
		<li>Cut open the lateral side of the knee and expose the two tendons. Cut the proximal tendon, leaving as much of the tendon as possible still attached to the muscle, and lift the EDL muscle (medial muscle) by gently pulling the tendon. Cut the distal tendon once it is exposed.</li>
	</ol>
	</li>
	<li>Mounting the muscle
	<ol>
		<li>Pin down the muscle via the tendons, and trim all the extra fat, fascia and tendon away as much as possible. Insert one tendon into a pre-tied knot and tie the suture tightly with suture tying forceps. Tie the second knot on around the metal hook.</li>
		<li>Repeat the same procedure with the long hook on the other end of the tendon. Make sure that none of the body of the muscle is contacted by the sutures. This will damage the preparation.</li>
		<li>Attach the short hook to the bottom of the experimental chamber and the long hook to the dual mode force transducer/motor. Bubble the solution in the experimental chamber with 100% oxygen.</li>
	</ol>
	</li>
	<li>Optimizing stimulation protocols and muscle length
	<ol>
		<li>Stretch the muscle by adjusting the micromanipulators attached to the transducer/motor to generate a baseline tension between 15 to 20 mN before finding the best stimulus parameters. Set the stimulation voltage to 40 V. The stimulation current is systematically increased until there is no additional increase in twitch force. The highest current found is increased by about 50% to ensure supra-maximal activation.</li>
		<li>Find the optimal length, L0, defined as the muscle length that give maximum twitch force, by increasing the muscle length and activating the muscle with a single twitch until the active force (peak force minus baseline force) stops increasing.</li>
		<li>Perform a short tetanic contraction (1 s activation) to test the mounting and stretch the muscle back to optimal baseline force if necessary. Record the muscle length in mm with a digital caliper.</li>
	</ol>
	</li>
</ol>
3. X-ray Diffraction
NOTE: The following description is for X-ray diffraction experiments done using the small angle X-ray diffraction instrument on the BioCAT beamline 18ID at the Advanced Photon Source, Argonne National Laboratory but similar methods could be employed on other beamlines such as ID 02 at the ESRF (France) and BL40XU at SPring8 (Japan). Beamline 18ID is operated at a fixed X-ray beam energy of 12 keV (0.1033 nm wavelength) with an incident flux of ~1013 photons per second in the full beam.
<ol>
	<li>Choose a specimen to detector distance (camera length). Use a 1.8 m camera length for experiments examining the 2.7 nm actin and high order myosin reflections such as 2.8 nm meridional reflections. Use a 4-6 m camera for other experiments, where one is primarily interested in fine detail on the meridian and layer lines</li>
	<li>Optimizing the position of the sample in beam
	<ol>
		<li>Determine the beam position by using a piece of X-ray sensitive paper that produces a dark spot in response to X-rays (&#34;a burn&#34;). Then use a video cross-hair generator to create a cross-hair aligned with the burn mark on the paper or simply make a mark on the video screen with a marker pen.</li>
		<li>Use the BioCAT supplied graphical user interface to the sample positioner to move the muscle to be centered on the beam position. Oscillate the sample chamber at ~10-20 mm/s by moving the sample stage in order to spread the X-ray dose over the muscle during the exposure. Observe the sample as it moves to avoid large regions of fascia (contains collagen which will pollute the diffraction patterns) and to ensure that it stays illuminated during the entire path of its travel. 
		NOTE: The exact steps required in sections 3.3 and 3.4 to make the required settings and actions using the beamline-supplied graphical user interface will be beamline and detector specific. Ask beamline staff as to how to perform these operations.</li>
	</ol>
	</li>
	<li>Setting up the CCD (charge coupled device) detector for high resolution patterns from muscle in defined static states (resting, or during isometric contraction)
	<ol>
		<li>Set up the exposure time and exposure period in the graphical user interface to the control software. Take a dark background image before taking the exposure and repeat this procedure every 2 hours or after changing of exposure time to correct any drift in the detector readout electronics.</li>
		<li>Attenuate the X-ray beam to desired value for the exposure. Then take an image. It is not possible to take sequences of images with this detector. The CCD detector also needs several seconds to read out an individual image.</li>
	</ol>
	</li>
	<li>Setting up the pixel array detector for a time resolved experiment
	<ol>
		<li>Set up the number of images, exposure time, exposure period in the graphical user interface. The pixel array detector used here needs at least 1 ms to readout. The maximum frame frequency for photon counting detector is 500 Hz. Use the photon counting detector output signal to control the X-ray shutter.</li>
		<li>Attenuate the beam to the desired intensity. Arm the detector and wait for the trigger from the data acquisition system. Synchronize the mechanical and X-ray data by triggering them at the same time. The X-ray patterns are collected continuously throughout the protocol a with a 1 ms exposure time and a 2 ms exposure period. 
		NOTE: The exact exposure time and exposure period should be determined on a case by case basis for the information desired and the observed lifetime of the sample in the beam. Attenuate the beam in order to use no more X-ray beam than is needed to provide analyzable data in the chosen exposure period.</li>
	</ol>
	</li>
</ol>
4. Post-experiment Muscle Treatment
<ol>
	<li>Recover and weigh the muscle after each mechanical and X-ray experiment. Calculate the cross-sectional area of the muscle using the measured muscle length and the muscle mass11 assuming a muscle density of 1.06 g/mL12.</li>
	<li>Stretch the muscle to the experimental length and fix the muscle in 10% formalin for 10 min. Separate the fixed muscle into a series of fiber bundles selected from locations throughout the entire muscle cross section3.</li>
	<li>Measure the sarcomere length using a video sarcomere length measuring system.</li>
</ol>

<ol><li>Ma, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thick-Filament+Extensibility+in+Intact+Skeletal+Muscle%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">Thick-Filament Extensibility in Intact Skeletal Muscle.</a> Biophysical Journal. 115 (8), 1580-1588 (2018).</li>
<li>Ma, W., Gong, H., Irving, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myosin+Head+Configurations+in+Resting+and+Contracting+Murine+Skeletal+Muscle%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">Myosin Head Configurations in Resting and Contracting Murine Skeletal Muscle.</a> International Journal of Molecular Sciences. 19 (9), (2018).</li>
<li>Kiss, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nebulin+stiffens+the+thin+filament+and+augments+cross-bridge+interaction+in+skeletal+muscle%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Nebulin stiffens the thin filament and augments cross-bridge interaction in skeletal muscle.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (41), 10369-10374 (2018).</li>
<li>Ochala, J., Gokhin, D. S., Iwamoto, H., Fowler, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pointed-end+capping+by+tropomodulin+modulates+actomyosin+crossbridge+formation+in+skeletal+muscle+fibers%5BTitle%5D)+AND+%22Federation+of+American+Societies+for+Experimental+Biology+Journal%22%5BJournal%5D)">Pointed-end capping by tropomodulin modulates actomyosin crossbridge formation in skeletal muscle fibers.</a> Federation of American Societies for Experimental Biology Journal. 28 (1), 408-415 (2014).</li>
<li>Lindqvist, J., Iwamoto, H., Blanco, G., Ochala, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+fraction+of+strongly+bound+cross-bridges+is+increased+in+mice+that+carry+the+myopathy-linked+myosin+heavy+chain+mutation+MYH4%28L342Q%29%5BTitle%5D)+AND+%22Disease+Models+%26+Mechanisms%22%5BJournal%5D)">The fraction of strongly bound cross-bridges is increased in mice that carry the myopathy-linked myosin heavy chain mutation MYH4(L342Q).</a> Disease Models & Mechanisms. 6 (3), 834-840 (2013).</li>
<li>Huxley, H. E., Stewart, A., Sosa, H., Irving, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((X-ray+diffraction+measurements+of+the+extensibility+of+actin+and+myosin+filaments+in+contracting+muscle%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">X-ray diffraction measurements of the extensibility of actin and myosin filaments in contracting muscle.</a> Biophysical Journal. 67 (6), 2411-2421 (1994).</li>
<li>Wakabayashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((X-ray+diffraction+evidence+for+the+extensibility+of+actin+and+myosin+filaments+during+muscle+contraction%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">X-ray diffraction evidence for the extensibility of actin and myosin filaments during muscle contraction.</a> Biophysical Journal. 67 (6), 2422-2435 (1994).</li>
<li>Anderson, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mavacamten+stabilizes+a+folded-back+sequestered+super-relaxed+state+of+%CE%B2-cardiac+myosin%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Mavacamten stabilizes a folded-back sequestered super-relaxed state of β-cardiac myosin.</a> Proceedings of the National Academy of Sciences of the United States of America. , (2018).</li>
<li>National Research Council. Committee for the Update of the Guide for the Care and Use of Laboratory Animals. Guide for the care and use of laboratory animals. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Institute+for+Laboratory+Animal+Research+%28U.S.%29+%26+National+Academies+Press+%28U.S.%29%22">Institute for Laboratory Animal Research (U.S.) & National Academies Press (U.S.)</a>. , National Academies Press. (2011).</li>
<li>Rand, C. How to Calibrate Your Dual-Mode Lever System Using DMC. , Available from: 
 <a target="_blank" href="https://aurorascientific.com/how-to-calibrate-your-dual-mode-lever-system-using-dmc/">https://aurorascientific.com/how-to-calibrate-your-dual-mode-lever-system-using-dmc/</a> (2017).</li>
<li>Alexander, R. M. V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+dimensions+of+knee+and+ankle+muscles+and+the+forces+they+exert%5BTitle%5D)+AND+%22Journal+of+Human+Movement+Studies%22%5BJournal%5D)">The dimensions of knee and ankle muscles and the forces they exert.</a> Journal of Human Movement Studies. 1, 115-123 (1975).</li>
<li>Burkholder, T. J., Fingado, B., Baron, S., Lieber, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Relationship+between+Muscle-Fiber+Types+and+Sizes+and+Muscle+Architectural+Properties+in+the+Mouse+Hindlimb%5BTitle%5D)+AND+%22Journal+of+Morphology%22%5BJournal%5D)">Relationship between Muscle-Fiber Types and Sizes and Muscle Architectural Properties in the Mouse Hindlimb.</a> Journal of Morphology. 221 (2), 177-190 (1994).</li>
<li>Jiratrakanvong, J., et al. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aJiratrakanvong%2C+J+%22MuscleX%3A+software+suite+for+diffraction+X-ray+imaging+V1.13.1%22">MuscleX: software suite for diffraction X-ray imaging V1.13.1</a>. , (2018).</li>
<li>Reconditi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myosin+filament+activation+in+the+heart+is+tuned+to+the+mechanical+task%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Myosin filament activation in the heart is tuned to the mechanical task.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (12), 3240-3245 (2017).</li>
</ol>

The authors declare that they have no competing financial interests.

Recent publications from our group showed that X-ray patterns from the mouse skeletal muscle can be used to shed light on sarcomeric structural information from muscle in health and disease1,2,3 especially with the increased availability of genetic modified mouse models for various myopathies. High resolution mechanical studies on single fibers or small bundles combined with X-ray diffraction is best done by experts. If, however, more modest mechanical information will suffice for your purposes, the whole muscle preparation allows collection of detailed X-ray patterns from a simple preparation.
A clean dissection is key to a successful combined mechanical and X-ray experiment. It is very important not to pull on the target muscle as well as other muscles associated with the soleus or EDL muscles during dissection since this could tear parts of the muscle and lead to reduced force. It can also lead to damaged internal structure that will degrade the X-ray patterns. Since everything will scatter in the X-ray beam, it is important to cleaning away any extra fat, the collagen in fascia as well as any hairs or loose bits of tissue while doing the following protocol. To reduce additional compliance in the muscle preparation, it is also important to securely tie the tendons to the hooks, as close as possible to the muscle body without damaging it.
Different X-ray exposure times can provide different kinds of information from the same muscle. Using the full beam on 18ID, an analyzable equatorial pattern can be obtained in a 1 ms exposure (See Figure 2D). For an analyzable first myosin layer line reflection, a 10 ms total exposure time is typically required. To collect higher order meridional reflections such as the M15 (2.8 nm myosin meridional reflection) and the 2.7 nm actin meridional reflection, typically at least 1 s total exposure is required but more than 2 s total exposure is recommended for high accuracy measurements.
The choice of the optimal X-ray detector for the experiment is important. For the most detailed X-ray patterns a customized CCD detector, such as the one at BioCAT with ca. 40 &#181;m pixels and ~65 &#181;m point spread functions in the phosphor, can provide patterns with high dynamic range and good spatial resolution but can only take one frame at a time. For time resolved experiments, the photon counting pixel array detector at BioCAT can collect X-ray patterns at 500 Hz. The 172 &#181;m pixel size with this detector, however, does not provide sufficient spatial resolution for detailed studies of the inner part of the meridian but is adequate for most other purposes. BioCAT acquired a high-resolution photon counting detector providing 75 &#181;m real resolution at maximum frame rate of 9,000 Hz. Similar detectors of this type are expected to supplant current detectors for muscle studies over the next few years.
With the very high fluxes of X-rays at third generation synchrotrons, radiation damage is a serious concern. It is always a good choice to attenuate the beam to deliver no more beam than is needed to observe the desired diffraction features. The same total X-ray exposure can be achieved by prolonging the exposure time from an attenuated beam. An advantage of photon counting pixel array detectors is that individual frames can be summed together with no noise penalty. Even then, radiation damage is possible. Signs of radiation damage includes drop of maximum force of contraction, smearing of layer line reflections, even change of muscle color.
One of the limitations of the intact mouse skeletal muscle preparation is the difficulty in obtaining sarcomere length from the intact muscle during the experiments. The muscles are too thick for video microscopy and laser diffraction. While with future developments it may be possible to estimate sarcomere length directly from the diffraction patterns14, in the near term the only option is to measure it after the experiment as described here.

Synchrotron small-angle X-ray diffraction is the method of choice for studying the nm-scale structure of actively contracting muscle preparations under physiological conditions. Importantly, structural information from living or skinned muscle preparations can be obtained in synchrony with physiological data, such as muscle force and length changes. There has been increasing interest in applying this technique to study the structural basis of inherited muscle diseases that have their basis in point mutations in sarcomeric proteins. The muscle biophysics community has been very active in generating transgenic mouse models for these human disease conditions that could provide ideal test beds for structural studies. Recent publications from our group1,2,3 and others4,5 have indicated that the X-ray patterns from the mouse extensor digitorum longus (EDL) and soleus muscles can provide all the diffraction information available from more traditional model organisms such as frog and rabbit psoas skeletal muscle. An advantage of the mouse skeletal muscle preparation is the ease of dissection and performing basic membrane-intact, whole muscle physiological experiments. The dimensions of the dissected muscle have sufficient mass to yield highly detailed muscle patterns in very short X-ray exposure times (~millisecond per frame) on third generation X-ray beamlines.
Muscle X-ray diffraction patterns consist of the equatorial reflections, the meridional reflections as well as the layer line reflections. The equatorial intensity ratio (ratio of the intensity of the 1,1 and 1,0 equatorial reflections, I11/I10), is closely correlated to the number of attached cross-bridges, which is proportional to the force generated in mouse skeletal muscle2. The meridional reflections that report periodicities within the thick and thin filaments can be used to estimate filament extensibility1,3,6,7. Diffraction features not on the meridian and the equator are called layer lines, which arise from the approximately helically ordered myosin heads on the surface of thick filament backbone as well as the approximately helically ordered thin filaments. The intensity of myosin layer lines is closely related to the degree of ordering of myosin heads under various conditions2,8. All of this information can be used study the behaviors of sarcomeric proteins in situ in health and disease.
Synchrotron X-ray diffraction of muscle has been historically done by teams of highly specialized experts but advances in technology and the availability of new data reduction tools indicate that this need not always be the case. The BioCAT Beamline 18ID at the Advanced Photon Source, Argonne National Laboratory has dedicated staff and support facilities for performing muscle X-ray diffraction experiments that can help newcomers to the field get started in using these techniques. Many users choose to formally collaborate with BioCAT staff, but an increasing number of users find they can do the experiments and analysis themselves reducing the burden on beamline staff. The primary goal of this paper is to provide training that provides potential experimenters with the information they need to plan and execute experiments on the mouse skeletal muscle system either at the BioCAT beamline or at other high flux beamlines around the world where these experiments would be possible.

<table><tbody><tr><td>#5 forceps</td><td>WPI</td><td>500342</td><td></td></tr><tr><td>4/0 surgical suture</td><td>Braintree Sci</td><td>SUT-S 108</td><td></td></tr><tr><td>Aquarium air stone</td><td>uxcell</td><td></td><td>a regular air stone from a pet store would be fine</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C5670</td><td></td></tr><tr><td>CCD detector</td><td>Rayonix Inc</td><td>MAR 165 CCD</td><td></td></tr><tr><td>Data acquisition system</td><td>Aurora Scientific Inc</td><td>610A</td><td></td></tr><tr><td>Elastomer compound</td><td>Dow Corning</td><td>Sylgard 184</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>High resolution photon counting detector</td><td>Dectris Inc</td><td>EIGER X 500K</td><td></td></tr><tr><td>High-power bi-phasic current stimulator</td><td>Aurora Scientific Inc</td><td>701</td><td></td></tr><tr><td>Iris Scissors</td><td>WPI</td><td>501263-G</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9541</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M7506</td><td></td></tr><tr><td>Micro scissor</td><td>WPI</td><td>503365</td><td></td></tr><tr><td>Motor/force transducer</td><td>Aurora Scientific Inc</td><td>300C-LR</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>Petri dish</td><td>Sigma-Aldrich</td><td>CLS430167</td><td></td></tr><tr><td>Photon counting detector</td><td>Dectris Inc</td><td>Pilatus 3 1M</td><td></td></tr><tr><td>Stainless Steel wire</td><td>McMaster-carr</td><td>8908K21</td><td></td></tr><tr><td>Suture Tying Forceps</td><td>WPI</td><td>504498</td><td></td></tr><tr><td>Video sarcomere length measuring system</td><td>Aurora Scientific Inc</td><td>900B</td><td></td></tr></tbody></table>

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.

Isometric tetanic contraction. Any kind of classic muscle mechanical experiment, such as isometric or isotonic contractions, can be performed with simultaneous acquisition of X-ray patterns. Figure 1A shows the experimental setup for mechanical and X-ray experiments. An example force trace for an isometric tetanic contraction is shown in Figure 1B. The muscle was held at resting for 0.5 s before activated for 1 s. The mechanical recording stops 1 s after the stimulus. The X-ray patterns were collected continuously throughout the protocol at 1 ms exposure time at 500 Hz.
X-ray diffraction patterns. The muscle X-ray diffraction pattern can give nanometer resolution structural information from structures inside the sarcomere. Muscle X-ray diffraction patterns are composed of four equivalent quadrants divided by the equator and the meridian. The equatorial pattern arises from the myofilament packing within the sarcomere perpendicular to the fiber axis, while the meridional patterns report structural information from the myofilaments along the muscle axis. The remaining reflections not on the equator or the meridian are called layer lines. Layer lines (e.g., features labeled MLL4 and ALL6 in Figure 2A) arise from the approximately-helical arrangement of molecular subunits within the myosin containing thick filaments and the actin containing thin filaments. The myosin-based layer lines are strong and sharp in patterns from resting muscle (Figure 2A), while actin-based layer lines are more prominent in patterns from contracting muscle (Figure 2B). Difference patterns obtained by subtracting the resting pattern from the contracting pattern (Figure 2C) can shed light on structural changes during force development in healthy and diseased muscle. By following these structural changes at the millisecond time scale of the molecular events during muscle contraction, the X-ray diffraction patterns can reveal substantial structural information (Figure 2D).
Data Analysis using MuscleX. Here is an example of equatorial reflections analysis using the &#8220;equator&#8221; routine in the MuscleX package (Figure 3). MuscleX is an open-source analysis software package developed at BioCAT13. The equatorial intensity ratio (I1,1/I1,0) is an indicator of the proximity of myosin to actin in resting muscle (Figure 3A), while it is closely correlated to the number of attached cross-bridges in contracting (Figure 3B) murine skeletal muscle2. The intensity ratio, I1,1/I1,0, is about 0.47 in resting muscle and about 1.2 in contracting muscle. The distance between the two 1,0 reflection (2*S1,0) is inversely related to the inter-filament spacing. Detailed documentations and manuals for MuscleX are available online13.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59559/59559fig1.jpg" /> 
Figure 1: Mechanical and X-ray experiment setup and protocol. (A) The muscle is mounted on one end to a hook inside the experimental chamber and the other end to a dual mode motor/force transducer. It is held between two Kapton film windows to allow the X-rays to pass through. The chamber is filled with Ringer&#8217;s solution perfused with 100% oxygen throughout the experiment. (B) The mechanical protocol for X-ray experiments on a muscle during tetanic contraction. <a href="https://www.jove.com/files/ftp_upload/59559/59559fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59559/59559fig2.jpg" /> 
Figure 2: EDL X-ray diffraction patterns. EDL muscle X-ray diffraction pattern from resting (A) and contracting (B) muscle. (C) The difference pattern between resting and contracting pattern. The blue region indicates high intensity in resting pattern, while the yellow region represents high intensity in contracting pattern. (D) X-ray diffraction pattern from a 1 ms exposure with EDL muscle. MLL1 = First order myosin layer line; MLL4 = Fourth order myosin layer line; ALL1 = First order actin layer line ALL6 = Sixth order actin layer line; ALL7 =&#160;Seventh order actin layer line; Tm = tropomyosin reflection (indicated by a white box); M3 = third order meridional reflection; M6 = sixth order meridional reflection. <a href="https://www.jove.com/files/ftp_upload/59559/59559fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59559/59559fig3.jpg" /> 
Figure 3: Data analysis of equatorial patterns using MuscleX. The background subtracted equatorial intensity ratio profile (while area) and first five orders (green lines) were fit to calculate the intensity of each peak. <a href="https://www.jove.com/files/ftp_upload/59559/59559fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about X-ray Diffraction of Intact Murine Skeletal Muscle as a Tool for Studying the Structural Basis of Muscle Disease at JoVE.com

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

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 ...

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Cell Biology

Developmental Biology

Unit 1

The Cytoskeleton I: Actin and Microfilaments

Analyzing Cells and Proteins

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7.2K Views.  Illinois Institute of Technology. Fysiologisch intacte muis skeletspier kan produceren hoge kwaliteit X-ray diffractie patronen met veel structurele informatie die inzicht kan geven in fysiologische processen. Röntgendiffractie is de enige techniek die het mogelijk maakt structurele informatie met hoge resolutie uit levend spierweefsel onder echte fysiologische omstandigheden in echte fysiologische tijd te verkrijgen. Veel spierziekten worden geërfd.Met een verhoogde beschikbaarheid om de meeste modellen van myopathi...

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

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

Medicine

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p &#62; 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.

Immunohistochemical staining of myosin heavy chain isoforms has emerged as the state-of-the-art discriminator of skeletal muscle fiber-type (i.e., type I, type IIA, type IIX, type IIB). Here, we present a staining protocol along with a novel semi-automated algorithm that facilitates rapid assessment of fiber-type and fiber morphology.

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of ...

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. ...

Biology

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...

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 ...

HE Staining and Micro-CT to Evaluate Murine Trabecular Bone Microarchitecture

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton is crucial for gaining insight into the pathophysiology of osteoporosis and improving its treatment. However, handling bone samples can be complex due to their hard and dense properties. Secondly, specialized software makes image processing and analysis difficult. In this protocol, we present a cost-effective and easy-to-use solution for trabecular bone microstructure analysis. Detailed steps and precautions are provided. Micro-CT is a non-destructive three-dimensional (3D) imaging technique that provides high-resolution images of trabecular bone structure. It allows for the objective and quantitative evaluation of bone quality, which is why it is widely regarded as the gold standard method for bone quality assessment. However, histomorphometry remains indispensable as it offers crucial cellular-level parameters, bridging the gap between two-dimensional (2D) and 3D assessments of bone specimens. As for the histologic techniques, we chose to decalcify the bone tissue and then perform traditional paraffin embedding. In summary, combining these two methods can provide more comprehensive and accurate information on bone microstructure.

Author Spotlight: An Economic and Efficient Method for Quantitative Evaluation of Bone Microarchitecture in a Murine Osteoporosis Model 

This protocol presents an economical and efficient method for quantitative evaluation of bone microarchitecture in a mouse model of osteoporosis by combining Hematoxylin-Eosin (HE) staining and micro-computed tomography (Micro-CT) techniques.

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton ...

Immunolabelling Myofiber Degeneration in Muscle Biopsies

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. Therapeutic options addressing the causes of muscular dystrophy pathogenesis are expected to alleviate muscle degeneration. Therefore, a method to assay and quantify the extent of cell death in muscle biopsies is needed. Conventional methods to observe myofiber degeneration in situ are either poorly quantitative or rely on the injection of vital dyes. In this article, an immunofluorescence protocol is described that stains necrotic myofibers by targeting immunoglobulin G (IgG) uptake by myofibers. The IgG uptake method is based on cell features characterizing the necrotic demise, including 1) the loss of plasma membrane integrity with the release of damage-associated molecular patterns and 2) the uptake of plasmatic proteins. In murine cross-sections, the co-immunolabelling of myofibers, extracellular matrix proteins, and mouse IgG allows clean and straightforward identification of myofibers with necrotic fate. This simple method is suitable for quantitative analysis and applicable to all species, including human samples, and does not require the injection of vital dye. The staining of necrotic myofibers by IgG uptake can also be paired with other co-immunolabelling.

Described here is a protocol for direct immunolabelling of necrotic myofibers in muscle cryosections. Necrotic cells are permeable to serum proteins, including immunoglobulin G (IgG). Revealing the uptake of IgG by myofibers allows the identification and quantification of myofibers that undergo necrosis regardless of muscle condition.

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. ...

Enzymatic Isolation and Characterization of Variable Length Mouse Muscle Fibers

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.

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).

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

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

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