This work was supported by the Paul D. Wellstone Cooperative Research Center (AR052646).

All procedures have been reviewed and approved by University of Pennsylvania IACUC.
1. EDL Dissection and Preparation (Approximately 30 min)
<ol>
	<li>Anesthetize mice to ensure that they experience no pain or distress during the procedure, but that the muscles remain well oxygenated by the circulation. We routinely use a Ketamine/xylazine cocktail (100/10 mg/kg) injected via IP. Surgical plane of anesthesia is determined by complete absence of pedal or palpedral withdrawal reflexes, or lack of ear twitch response.</li>
	<li>Immobilize the hindlimbs using medical tape, and remove the skin of the lower anterior hindlimb to expose the muscles of this area. Keep the muscles moist with the application of PBS at regular intervals.</li>
	<li>Under a dissecting scope, make a small incision lateral to the knee in order to expose the proximal tendon of the EDL muscle. There are two tendons in this region, and both should be cut to enable removal of the EDL.</li>
	<li>At the medial ankle, cut the tendon of the TA muscle to expose the distal tendons of the EDL, which extend along the meta tarsals. Lift the TA muscle out of the way, being careful not to cut or touch the EDL underneath. Return to the distal tendons of the EDL, snip each one and draw them through the ankle, then grasping the tendons, gently pull the EDL away from the rest of the limb. It should release from the proximal end freely, but if not, return to the lateral incision to ensure that the tendon is cut. Remove the muscle and place in a dissecting dish filled with chilled oxygenated Ringers. Pin the muscle through the tendons at approximately resting length, which is the length found in vivo. The length is too short when the muscle is flaccid in the dish, and too long if the muscle is pulling on the dissecting pins.</li>
	<li>Euthanize the mouse immediately following muscle removal by cervical dislocation.</li>
	<li>Tie sutures to the tendons, as close as possible to the muscle, but not touching the muscle. Pin the muscle at approximately resting length using the sutures.</li>
</ol>
2. Diaphragm Dissection and Preparation (Approximately 30 min)
<ol>
	<li>Euthanize the mice prior to this procedure by cervical dislocation under anesthesia, if the same mouse is subjected to both dissections, or by CO2 followed by confirmation with cervical dislocation. </li>
	<li>Make an incision in the skin to expose the abdominal and chest cavity. Open the abdominal cavity, and cut the body wall just below the ribs. Using bone scissors, and starting above the diaphragm insertion, cut around the entire rib cage following the line of the ribs, and cut through spine. Cut blood vessels running through the center of the diaphragm so that the diaphragm can be removed easily.</li>
	<li>Remove the diaphragm from the mouse, and place in a dissecting dish filled with oxygenated Ringers. Gently agitate the diaphragm in the dish to wash away blood. Refresh Ringers solution as needed.</li>
	<li>Cut a small strip of the diaphragm from the central tendon to the ribs along the orientation of the fibers in the central portion of the lateral hemidiaphragms. The strip should be between 2-4 mm wide. Using bone scissors, cut the rib on either side of the strip, leaving approximately 1-2 mm overhang of rib on either side of the diaphragm strip.</li>
	<li>Tie sutures to the central tendon. Tie suture to each of the laterally protruding rib ends, and then tie these together to make a large loop.</li>
	<li>Two strips can be obtained from any given diaphragm (one from each side).</li>
</ol>
3. Mounting Muscles in Mechanics Bath
<ol>
	<li>Grasp the sutures and use these to attach the muscle to a rigid post on one end, and to a force transducer on the other end.</li>
	<li>Adjust the length to make sure the muscle is not slack, but isn't taut. A good approximation is the resting muscle length as in 1.4 - 1.5.</li>
	<li>Bath should be filled with oxygenated Ringers solution maintained at 22 &deg;C to prolong muscle stability for testing. Muscles should rest for 5 min in this bath prior to functional testing so that muscle temperature is equilibrated to 22 &deg;C.</li>
</ol>
4. Isometric Contractions
<ol>
	<li>Establish Supramaximal Stimulation Conditions. After a muscle is placed in the bath, use single 0.5 msec stimulation pulses to generate a twitch, and monitor force output. Gradually increase current until the force attains a maximum but steady level. Increase current to 10% more than this level for the remaining experiments.</li>
	<li>Establish Optimum Length. Using isometric twitch stimulations, adjust the length of the muscle gradually until a maximal force is obtained. Rest the muscle ~10 sec between each contraction. Optimal muscle length (Lo) is achieved when twitch force is maximal. Record muscle length (the length between the myotendinous junctions for EDL, and between the central tendon myotendinous junction and the muscle insertion to the rib) using Vernier calipers.</li>
	<li>Maximum Isometric Tetanic Force. Stimulate muscle set at Lo for a period of 500 msec, with a series of 0.5 msec pulses at supramaximal stimulation and at fusion frequency (from the plateau of the frequency-force relationship (e.g. Ref: 8). The plateau for EDL muscles is typically achieved with 120 Hz, and for diaphragm muscles with 100 Hz. Stimulate 3 times at the respective frequency with rest periods of 5 min between stimulation bouts for every muscle.</li>
</ol>
5. Eccentric Contractions
<ol>
	<li>Allow muscle to rest 5 min between performing isometric and eccentric contractions.</li>
	<li>Stimulate muscles at 80 Hz isometrically for the initial 500 msec, followed by a 10% Lo stretch in the final 200 msec stimulation (a stretch rate of 0.5 Lo/sec).</li>
	<li>Repeat stimulation pattern with 5 min rests between for the desired number of eccentric contractions.</li>
	<li>Measure force for each contraction in the time period prior to the stretch. Calculate the drop in force between the first and last contraction.</li>
</ol>
6. Muscle Removal from the Apparatus
<ol>
	<li>Shorten the muscle length to allow for gentle removal of the muscle from the transducer and post and return it to a dissecting dish with Ringers.</li>
	<li>Remove sutures from the muscles.</li>
	<li>For the EDL muscle, blot the muscle twice, then weigh it, before subsequent processing (e.g. freezing, fixing, etc). This will be important for calculating the cross sectional area, and specific force.</li>
	<li>For the diaphragm muscle, soak in 0.1% procion orange, a membrane impermeant dye for 15-20 min. This will provide an index of dissection damage.</li>
	<li>Dissect the muscle away from the bony insertion, as well as the central tendon. This is necessary to provide an accurate weight of the muscle. Blot as above before weighing and subsequent preparation.</li>
	<li>Calculate Cross Sectional Area (CSA): 
	CSA (mm2) = mass (mg) / [(Lo mm) * (L/Lo) * (1.06 mg/mm3)], 
	where L/Lo is the fiber to muscle length ratio (0.45 for EDL, 1.0 for the diaphragm)5 and 1.06 is the density of muscle.</li>
</ol>

<ol>
	<li>Barton, E. R., Khurana, T. S., Lynch, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+isometric+force+of+isolated+mouse+muscles+in+vitro.">Measuring isometric force of isolated mouse muscles in vitro.</a> TREAT-NMD. , (2008).</li><li>Barton, E. R., Morris, L., Musaro, A., Rosenthal, N., Sweeney, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle-specific+expression+of+insulin-like+growth+factor+I+counters+muscle+decline+in+mdx+mice.">Muscle-specific expression of insulin-like growth factor I counters muscle decline in mdx mice.</a> J. Cell Biol. 157, 137-148 (2002).</li><li>Barton, E. R., Morris, L., Kawana, M., Bish, L. T., Toursel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+administration+of+L-arginine+benefits+mdx+skeletal+muscle+function.">Systemic administration of L-arginine benefits mdx skeletal muscle function.</a> Muscle Nerve. 32, 751-760 (2005).</li><li>Barton, E. R., Wang, B. J., Brisson, B. K., Sweeney, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diaphragm+displays+early+and+progressive+functional+deficits+in+dysferlin-deficient+mice.">Diaphragm displays early and progressive functional deficits in dysferlin-deficient mice.</a> Muscle Nerve. 42 (1), 22-229 (2010).</li><li>Brooks, S. V., Faulkner, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+properties+of+skeletal+muscles+from+young,+adult+and+aged+mice.">Contractile properties of skeletal muscles from young, adult and aged mice.</a> J. Physiol. 404, 71-82 (1988).</li><li>Harcourt, L. J., Schertzer, J. D., Ryall, J. G., Lynch, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+dose+formoterol+administration+improves+muscle+function+in+dystrophic+mdx+mice+without+increasing+fatigue.">Low dose formoterol administration improves muscle function in dystrophic mdx mice without increasing fatigue.</a> Neuromuscul. Disord. 17 (1), 47-55 (2007).</li><li>Lynch, G. S., Hinkle, R. T., Chamberlain, J. S., Brooks, S. V., Faulkner, J. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dystrophin+protects+the+sarcolemma+from+stresses+developed+during+muscle+contraction.">Dystrophin protects the sarcolemma from stresses developed during muscle contraction.</a> Proc. Natl. Acad. Sci. U.S.A. 90, 3710-3714 (1993).</li><li>Salomonsson, S., Grundtman, C., Zhang, S. J., Lanner, J. T., Li, C., Katz, A., Wedderburn, L. R., Nagaraju, K., Lundberg, I. E., Westerblad, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upregulation+of+MHC+class+I+in+transgenic+mice+results+in+reduced+force-generating+capacity+in+slow-twitch+muscle.">Upregulation of MHC class I in transgenic mice results in reduced force-generating capacity in slow-twitch muscle.</a> Muscle Nerve. 39 (5), 674-6782 (2009).</li><li>Stedman, H. H., Sweeney, H. L., Shrager, J. B., Maguire, H. C., Panettieri, R. A., Petrof, B., Narusawa, M., Leferovich, J. M., Sladky, J. T., Kelly, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mdx+mouse+diaphragm+reproduces+the+degenerative+changes+of+Duchenne+muscular+dystrophy.">The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy.</a> Nature. 352, 536-539 (1991).</li><li>Welch, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTC124+targets+genetic+disorders+caused+by+nonsense+mutations.">PTC124 targets genetic disorders caused by nonsense mutations.</a> Nature. 447 (7140), 87-91 (2007).</li></ol>

Production and Free Access to this article is sponsored by Aurora Scientific.

The goal of this article is to provide guidance for performing isolated muscle function on two muscles from mice - the EDL and the diaphragm. Evaluation of these muscles can provide insight into whether or not therapeutic candidates for muscle disease are beneficial. For both muscles, the major factor in obtaining robust data is a clean dissection. Therefore, practicing and perfecting the initial isolation step is essential before moving onto functional testing. In addition, establishing functional benchmarks for normal muscle is critical prior to making comparisons to dystrophic muscle, or between treatments. This will assure that the study results are not subject to high variability imparted by the ability of the person performing the experiments. The use of a membrane impermeant dye can help with optimizing the dissection preparation, for incubation of any muscle in a solution containing such a dye will mark most damaged fibers, and can serve as an index of dissection success. Minimizing the number of fibers damaged in the muscle will help to optimize the measurement. The fibers damaged by dissection fluoresce much more brightly than those damaged by eccentric contraction, and so if the dye is also used during the mechanics process, one can use the intensity of dye to distinguish between the two types of damage.
Dissection of diaphragm strips will almost always have fiber damage, simply because by cutting along the length of the fibers, some inevitably get destroyed. Dye uptake is strong in those damaged fibers, and these are normally restricted to the outer edges of the preparation. On average, we observe a band of damaged fibers that is ~3 fibers in width (~120 &mu;m) on either side of the muscle strip. If the damaged band comprises more than 15% of the muscle, then the data is discarded. The diaphragm preparation also has constraints on the optimum size for functional measures. We have found that pieces of diaphragm that are wider than 5 mm begin to fold up on themselves, because the central tendon tie is only at one point. This results in decreased specific force in the preparation. We have also found that narrower strips also have lower specific force, which we believe is because the numbers of fibers damaged during dissection comprise a larger proportion of the total fiber number. For instance, if 0.1 mm on each side is damaged, then that is 5% (2x0.1 mm / 4 mm strip) of the muscle preparation that doesn't contribute to force, whereas if the strip is only 1 mm, then 20% of the muscle preparation is damaged. Thus, both the width of the damaged region as well as the width of the entire preparation are important factors to control.
Measurements of maximum isometric tension require that all muscle fibers in a muscle are stimulated. Because there is tremendous variability in the components of a function apparatus, this must be determined for each individual set up. For example, bath size or the type of stimulator can affect the intensity of stimulation. Twitch stimulation is a reasonable way to determine supramaximal stimulation conditions. Once this is established for a specific setup and muscle, it can be utilized for subsequent studies.
In contrast, the optimum length of any given muscle needs to be measured for each preparation using the iterative process described above. This ensures that thick and thin filament overlap is optimal and the maximum potential force generating capacity is measured. Alternative procedures can rely on the diffraction patterns associated with the muscle striations, but this requires additional equipment not described here.
We routinely use 500 msec stimulation durations, which falls in the middle range of this parameter used by other investigators in this field. Although this might cause some fatigue of the muscle during the contraction, which is evident by a "sag" in the maximal force production, this in itself can be informative. For instance, a difference in fatigue could be achieved by different types of therapies including those that target calcium handling, hence the sag in force during active contraction can serve as an index for improvement. Alternatively, the loss of force could indicate that the sutures on the tendons are not tight enough, and that they are slipping during the contraction. The muscle must be removed from the bath and the sutures must be re-tied if this occurs. We also use a series of 3 tetanic contractions, which helps evaluate the stability of the preparation. Again, suture slips would lead to loss of force between contractions, requiring suture re-tying. Large muscles may also generate anoxic cores, which lead to losses of force during the protocol. Muscle size is a limiting factor for performing isolated muscle function testing, where EDL muscles with masses greater than 20 mg lose force with each contraction, and cannot be supported by superfusion in a bath. Diaphragm strips do not suffer from the same complications because they are thin enough to have prolonged viability in the bath.
Other muscles can be utilized for isolated muscle function, including the soleus muscle, which is commonly used, but not described here. Many of the same procedures can be adopted for the soleus in terms of the preparation and the functional testing. However, the main differences are in the stimulation frequency, and the parameters for eccentric contractions. Use of the soleus for function complements that of the other two muscles, and so it should be considered as part of a "standard package " for evaluating dystrophic mice 4, 7.
Eccentric contraction provides an index of contractile fragility, and it is important to use a protocol that results in modest loss of force in muscles from wildtype animals and a significant loss of force in untreated dystrophic muscles so that there is a wide dynamic range for comparisons. A lack of any force loss in normal muscles suggests that the eccentric contraction is too mild, and will not be adequate to distinguish between therapies that are not effective and those that are truly beneficial. However, a dramatic loss of force in normal muscles subjected to eccentric contraction may be too great to tease out differences associated with the disease. Our protocol for the EDL and diaphragm uses a 0.5 Lo/sec stretch rate to produce a 10% length change. We typically perform 5 eccentric contractions, which result in a small loss of force in normal muscles and a significant loss of force in dystrophic muscles. Certainly, all of these parameters can be varied to increase the overall length change, the rate of stretch, or the number of eccentric contractions in order to distinguish differences between diseased and healthy muscle, as well as on the effects of a specific type of mutation or treatment one is studying. As long as there is a marked difference between normal and diseased muscles, then there is a gold standard to reach for in terms of treatments.
In summary, this protocol establishes guidelines for performing isometric and eccentric contractions, and hopefully identifies the potential pitfalls to avoid when setting this technique up in your lab.

Muscle function measurements contribute to the evaluation of potential treatments for muscle pathology, as well as to the determination of mechanisms underlying physiology of this tissue. For muscle disease, the use of mouse models have become a central component for understanding the links between genotype and phenotype, and for extending that knowledge into the design and testing of potential therapeutics. The muscular dystrophies, in particular, have relied on mice to evaluate these agents and establish pre-clinical data required to move forward to trials in patients. A frequent outcome measure uses isolated muscle function to determine strength, which is applicable to a wide range of studies. Another measure is the use of eccentric, or lengthening, contractions to determine changes in muscle membrane integrity, which is deficient in Duchenne muscular dystrophy, and the mouse model for this disease (mdx). Therefore, it is essential for these types of measurements to be sensitive and reproducible.
The mouse extensor digitorum longus (EDL) muscle has been used extensively for isolated muscle function due to its ideal geometry and size, including uniform fiber orientation and definitive tendons 2, 5, 6, 10, 12. Methods for EDL muscles isometric functional measurements have been described in a previous JoVE publication 8, as well as in the Treat-NMD SOP 1. We have extended the description of these methods to include both isometric and eccentric contractions. Hallmarks of disease are evident in the EDL, including heighted cycles of degeneration/regeneration and diminished force output.
The mouse diaphragm exhibits the most rapid pathological progression of muscular dystrophy compared to other muscles in the mouse 11. By 6 months of age, cumulative fibrosis comprises approximately 50% of the muscle. This results in significantly impaired force output 11. Therefore, therapeutic agents that can prevent fibrotic infiltration can be evaluated efficiently in the diaphragm.
The loss of dystrophin in muscle leads to increased fragility and heightened contractile damage in all muscles 9. Therefore many therapies for Duchenne muscular dystrophy are geared for dystrophin replacement. As such, an assay that has become essential for evaluating these strategies is eccentric contraction, which can distinguish between normal and dystrophic muscle, as well as determine what benefit a particular strategy has for protecting a dystrophic muscle from contractile damage 2, 3, 4, 12. This procedure requires either a dual-mode servo-motor that can modulate/record length and force, or a method of adjusting length rapidly separate from a force transducer.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>in vitro Muscle Test System</td>
 <td>Aurora Scientific</td>
 <td>1200A</td>
 <td></td>
 </tr>
 <tr>
 <td>Dissecting microscope</td>
 <td>Leica</td>
 <td>MZ6</td>
 <td></td>
 </tr>
 <tr>
 <td>ACE light source</td>
 <td>Schott-Fostec</td>
 <td>A20500</td>
 <td></td>
 </tr>
 <tr>
 <td>Dissecting scissors</td>
 <td>Fine Science Tools</td>
 <td>14060-11</td>
 <td></td>
 </tr>
 <tr>
 <td>Angled dissecting scissors</td>
 <td>Fine Science Tools</td>
 <td>15006-09</td>
 <td></td>
 </tr>
 <tr>
 <td>Scalpel handle</td>
 <td>Fine Science Tools</td>
 <td>10003-12</td>
 <td>alternate dissecting tool</td>
 </tr>
 <tr>
 <td>Curved scalpel blades #12</td>
 <td>Fine Science Tools</td>
 <td>10012-00</td>
 <td>alternate dissecting tool</td>
 </tr>
 <tr>
 <td>Bone scissors</td>
 <td>Fine Science Tools</td>
 <td>16044-10</td>
 <td></td>
 </tr>
 <tr>
 <td>S&amp;T suture tying forceps</td>
 <td>Fine Science Tools</td>
 <td>00272-13</td>
 <td></td>
 </tr>
 <tr>
 <td>Dumont SS forceps - angled</td>
 <td>Fine Science Tools</td>
 <td>11203-25</td>
 <td></td>
 </tr>
 <tr>
 <td>Braided silk suture size 6-0</td>
 <td>Teleflex Medical</td>
 <td>07-30-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Medical tape</td>
 <td>Transpore</td>
 <td>3M</td>
 <td></td>
 </tr>
 <tr>
 <td>Ketamine hydrochloride 100 mg/ml</td>
 <td>Hospira</td>
 <td>NDC 0409-2051-05</td>
 <td>Final Dose is 80 mg/kg</td>
 </tr>
 <tr>
 <td>TranquiVed Injection (xylazine 100 mg/ml)</td>
 <td>Vedco</td>
 <td>NDC 50989-234-11</td>
 <td>Final Dose is 10 mg/kg</td>
 </tr>
 <tr>
 <td>Reactive orange 14</td>
 <td>Sigma-Aldrich</td>
 <td>R-8254</td>
 <td></td>
 </tr>
 <tr>
 <td>Ringers Solution Components</td>
 <td></td>
 <td></td>
 <td>Solution is gas equilibrated with 95% O2 and 5% CO2, final pH 7.4</td>
 </tr>
 <tr>
 <td>Sodium chloride</td>
 <td>Sigma-Aldrich</td>
 <td>S7653</td>
 <td>Final Concentration: 118 mM</td>
 </tr>
 <tr>
 <td>Potassium chloride</td>
 <td>Fisher Scientific</td>
 <td>P217-3</td>
 <td>Final Concentration: 4.7 mM</td>
 </tr>
 <tr>
 <td>Calcium chloride dihydrate</td>
 <td>Fisher Scientific</td>
 <td>C79-500</td>
 <td>Final Concentration: 2.5 mM</td>
 </tr>
 <tr>
 <td>Potassium phosphate monobasic</td>
 <td>Fisher Scientific</td>
 <td>P-285</td>
 <td>Final Concentration: 1.2 mM</td>
 </tr>
 <tr>
 <td>Magnesium sulfate</td>
 <td>J.T. Baker</td>
 <td>2500-01</td>
 <td>Final Concentration: 0.57 mM</td>
 </tr>
 <tr>
 <td>4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)</td>
 <td>Fisher Scientific</td>
 <td>BP310-500</td>
 <td>Final Concentration: 5.95 g/L</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>Sigma-Aldrich</td>
 <td>G8270</td>
 <td>Final Concentration: 5.5 mM</td>
 </tr>
 </tbody>
</table>

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.

Expected values for the isometric forces of EDL muscles from 12 week old animals are shown in Figure 1. Because mdx muscles exhibit compensatory hypertrophy, total tetanic force can be higher in mdx muscle compared to age matched wildtype controls. However, a more appropriate measure of functional output is specific force (Figure 2), where force is normalized for cross-sectional area to calculate this value. Specific force depends on the inherent functional capacity of the muscle, where the weakness in dystrophic muscles is more apparent when both absolute force and cross sectional area are accounted for. In our hands, EDL muscles from mdx mice generate approximately 20 - 25% lower specific forces than those of age matched wildtype mice from 10 - 26 weeks of age.
For the diaphragm, only specific force is relevant for comparisons because the preparation is a piece of the muscle dependent upon the dissection. Comparison of specific force between wildtype and mdx diaphragm muscles reflects the progressive pathology in this tissue. Isometric force output diminishes with age (Figure 3), so that by 6 months of age, diaphragm muscles from mdx mice produce no more than half of the functional output of diaphragm strips from age-matched wildtype controls.
An example of eccentric contractions from diaphragm testing is shown in Figure 4. With each subsequent eccentric contraction, force output diminishes in diaphragm strips from both wildtype (C57) and mdx mice. However, the loss of force is more dramatic in muscle samples from the mdx mouse, presumably from the absence of dystrophin and its associated proteins.
<img alt="Figure 1" src="/files/ftp_upload/50036/50036fig1.jpg" /> 
 Figure 1. Isometric tetanic force in wildtype and mdx EDL muscles from 12 week old mice. Absolute force can be higher in age-matched mdx EDL muscles due to compensatory hypertrophy.
<img alt="Figure 2" src="/files/ftp_upload/50036/50036fig2.jpg" /> 
 Figure 2. Isometric tetanic force normalized by muscle cross sectional area is Specific Force, and reveals depressed functional capacity of EDL muscles from mdx mice compared to those of wildtype mice. Traces are from the same mice as in Figure 1.
<img alt="Figure 3" src="/files/ftp_upload/50036/50036fig3.jpg" /> 
 Figure 3. The progressive loss of force generating capacity assessed by specific force is most apparent in the diaphragm of mdx mice (red bars) compared to wildtype (C57 in blue line).
<img alt="Figure 4" src="/files/ftp_upload/50036/50036fig4.jpg" /> 
 Figure 4. The loss of force as a function of eccentric contraction number for diaphragm muscles from 12 week old C57 and mdx mice. Data is mean &plusmn; SEM for N=4 muscles per genotype.

Watch this Scientific Journal Video about Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies at JoVE.com

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

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. ...

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JoVE Journal

Biology

32.5K Views.  School of Dental Medicine, University of Pennsylvania. 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 wi...

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

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3&#039;UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

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 ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been reported that skeletal muscle may increase the extraction of glucose from the blood by up to 50-fold during exercise compared to resting conditions. The increase in muscle glucose uptake during exercise and insulin stimulation is dependent on the translocation of glucose transporter 4 (GLUT4) from intracellular compartments to the muscle cell surface membrane, as well as phosphorylation of glucose to glucose-6-phosphate by hexokinase II. Isolation and incubation of mouse muscles such as m. soleus and m. extensor digitorum longus (EDL) is an appropriate ex vivo model to study the effects of insulin and electrically-induced contraction (a model for exercise) on glucose uptake in mature skeletal muscle. Thus, the ex vivo model permits evaluation of muscle insulin sensitivity and makes it possible to match muscle force production during contraction ensuring uniform recruitment of muscle fibers during measurements of muscle glucose uptake. Moreover, the described model is suitable for pharmacological compound testing that may have an impact on muscle insulin sensitivity or may be of help when trying to delineate the regulatory complexity of skeletal muscle glucose uptake.
Here we describe and provide a detailed protocol on how to measure insulin- and contraction-stimulated glucose uptake in isolated and incubated soleus and EDL muscle preparations from mice using radiolabeled [3H]2-deoxy-D-glucose and [14C]mannitol as an extracellular marker. This allows accurate assessment of glucose uptake in mature skeletal muscle in the absence of confounding factors that may interfere in the intact animal model. In addition, we provide information on metabolic viability of incubated mouse skeletal muscle suggesting that the method&#160;applied possesses some caveats under certain conditions when studying muscle energy metabolism.

Intact regulation of muscle glucose uptake is important for maintaining whole body glucose homeostasis. This protocol presents assessment of insulin- and contraction-stimulated glucose uptake in isolated and incubated mature skeletal muscle when delineating the impact of various physiological interventions on whole body glucose metabolism.

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been ...

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.

In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence analysis.

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in ...

In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig

Reliable assessment of skeletal muscle strength is arguably the most important outcome measure in neuromuscular and musculoskeletal disease and injury studies, particularly when evaluating regenerative therapies&#39; efficacy. Additionally, a critical aspect of translating many regenerative therapies is the demonstration of scalability and effectiveness in a large animal model. Various physiological preparations have been established to evaluate intrinsic muscle function properties in basic science studies, primarily in small animal models. The practices may be categorized as: in vitro (isolated fibers, fiber bundles, or whole muscle), in situ (muscle with intact vascularization and innervation but distal tendon attached to a force transducer), and in vivo (structures of the muscle or muscle unit remain intact). There are strengths and weaknesses to each of these preparations; however, a clear advantage of in vivo strength testing is the ability to perform repeated measurements in the same animal. Herein, the materials and methods to reliably assess isometric torque produced by the hindlimb dorsiflexor muscles in vivo in response to standard peroneal electrical stimulation in anesthetized pigs are presented.

In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig

The present protocol describes concise experimental details on the evaluation and interpretation of in vivo torque data obtained via electrical stimulation of the common peroneal nerve in anesthetized pigs.

Reliable assessment of skeletal muscle strength is arguably the most important outcome measure in neuromuscular and musculoskeletal disease and injury ...