This work was supported by AHA SDG 10SDG2630086 to Zhao X, RO1-AR061385 to Ma J and GO Grant RC2AR05896 to Brotto M.

Solution composition:
2.5 mM Ca2+ Tyrode solution: 140 mM NaCl, 5 mM KCl, 10 mM HEPES, 2.5 mM CaCl2, 2 mM MgCl2 and 10 mM glucose
0 mM Ca2+ Tyrode solution: 140 mM NaCl, 5 mM KCl, 10 mM HEPES, 2 mM MgCl2, 0.1 mM ethylene glycol tetraacetic acid (EGTA) and 10 mM glucose
Note: bathing solution should be saturated with 100% O2 if using the above solution, but with 95% O2</su...

<ol>
	<li>Winegrad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+intracellular+calcium+movements+in+excitation-contraction+coupling+in+skeletal+muscle.">Role of intracellular calcium movements in excitation-contraction coupling in skeletal muscle.</a> Fed. 24, 1146-1152 (1965).</li><li>Sandow, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitation-contraction+coupling+in+skeletal+muscle.">Excitation-contraction coupling in skeletal muscle.</a> Pharmacol. Rev. 17, 265-320 (1965).</li><li>Thornton, A. 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No conflicts of interest declared.

Measurement of contractile force and fatigability is important for the overall evaluation of skeletal muscle function. The major purpose of this assay is to identify changes in muscle force and fatiguing properties under certain pathological conditions, such as sarcopenia and muscle fatigue, and to test the effect of drugs/reagents on muscle contractility. Since the muscle force is closely related with intracellular Ca2+ release, extracellular Ca2+ entry and the crosstalk between these two, we can a...

Skeletal muscles attach to bones of the skeleton and generate contractile forces under the control of the central nervous system. Excitation-Contraction coupling (ECC) refers to the process of converting an electrical stimulus to a mechanical response. Ca2+ signaling is an essential component of the contractile function in skeletal muscle. Effective Ca2+ mobilization from sarcoplasmic reticulum (SR) is an important component for ECC in muscle cells1, 2, and changes in intracellular Ca2+ signaling underlie the corresponding contractile dysfunction in a number of muscle diseases3-5. Proper assessment of muscle contractility is essential and complimentary to Ca2+ imaging and other assays to gain insights into skeletal muscle function, not just at the contractile level, but also at the kinetic level. Force and speed can also be obtained to inform the important property of muscle power and the status of the ECC process under different physiological and pathophysiological conditions.
This fecund field of research has a very rich history and many theories of muscle contraction appeared over two millennia6. Modern muscle research probably begins in 1674-1682 with the microscopic observation of cross-striations and myofibrils in muscle fibers by Leeuwenhoek6. Almost a century later, Luigi Galvani observed that frog muscle contracts vigorously when its nerve is touched with scalpel during a spark discharge from a distant electric machine7-9. Contraction could also be produced by connecting the leg nerve to the muscle through a metal conductor. The details of the complex electrical signaling mechanism advocated by Galvani were eventually formulated by Hodgkin, Huxley and Katz in their famous equation10, 11 that became the foundation of electrophysiology. The remarkable observations of Ringer on the effects of extracellular Ca2+ on the contractility of frog heart and skeletal muscles12-15 represent the first major step in the recognition of Ca2+ as a key regulator of muscle contractility16, 17. From the 1980's to the present day a burst of discoveries in the muscle contractility field was realized due to the introduction of muscle contractility and fatigability protocols in murine skeletal muscles18. Jones and Edwards were the first to suggest that low frequency intermittent fatigue (exercise-induced reduction in force)19 was associated with changes in the ECC machinery and not the contractile apparatus. In the late 1980's and early 1990's, Kolkeck et al20, Kolbeck and Nosek 21, and Reid 22 were using diaphragm muscle from rodent models to study the effects of theophyllines, cortiosterone, and free radicals on skeletal muscle contractility, while Brooks and Faulkner were the first to report on measurements of repeated force and power measurements in fast- and slow-muscles from mice22. In addition, Lannegren, Westerblad, Lamb, and Westerblad were the first to directly link ex vivo contractility with intracellular Ca2+ regulation and started questioning the role of acidosis in muscle fatigue23, 24.
Our laboratories have significantly contributed since the early 2000's towards understanding of novel genes with modulatory and regulatory roles on muscle ECC with critical roles in muscle contractility, fatigability, and aging by using a combination of intact mouse muscle contractility studies, intracellular Ca2+ monitoring in intact and skinned muscle fibers and molecular-genetic manipulations3-5, 25-29.
Here we detailed the experimental protocol for measuring contractility of murine isolated soleus and extensor digitorum longus (EDL) muscles, which correspond to a mostly slow-oxidative (type I and IIa muscle fibers) and a mostly fast-glyocolytic muscle (type IIb and IIx muscle fibers) with distinct contractile properties. In this protocol, intact muscle-tendon complexes were isolated and bathed in an ADI PowerLab Radnotti chamber system supplied with either pure oxygen or a mixture of oxygen (95%) and CO2 (5%). Contractile forces were generated by electrical stimulations from a Grass stimulator and detected using a force transducer that was integrated with an ADI PowerLab/400 system, allowing customization of macro routines to control the acquisition, collection, digitization, and storage of data. This setup can measure muscle force, muscle power, as well as the force vs. frequency relationship, muscle fatigue, recovery from muscle fatigue, speed and overall kinetic properties of muscle contraction. In addition, the effects of drugs on muscle contraction can be monitored through these experiments.
Advantages of this method lay in removing the neuronal and vascular components away from the skeletal muscle, allowing direct assessment of the intrinsic properties of contracting muscle. In addition, ex vivo contractility assays allow manipulation of the extracellular milieu surrounding the isolated muscles, which enables the use of pharmacological manipulations of various ion permeation channels and transporters in order to define their physiological roles for skeletal muscle function.
This ex vivo system has allowed us to recently discover a distinct alternan behavior in certain mutant muscle preparations, which were linked to altered intracellular Ca2+ handling properties4. Alternans are defined as fluctuating burst episodes of contractile force during the decline phase of the fatiguing profile. During these events contractile forces momentarily increase above its previous level of force during fatiguing stimulation, perhaps because either more Ca2+ is being released or the contractile machinery has become more sensitive to Ca2+&nbsp;&nbsp;30. Treatment of cyclopiazonic acid (CPA), a reversible blocker of sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA), caffeine, an agonist of ryanodine channel (RyR) and repeated fatiguing stimulations can all induce mechanical alternans4, suggesting that alternans are directly related to modulation of the E-C coupling process. Demonstration of the method to induce and record mechanic alternans in in vitro contractility setup serves as an example to show the diversified experimental parameters that could be obtained with this system or similar ones, based on individual research interests.
This method may be of interest for researchers studying muscle physiology. Similar setup can also be used for isolated skeletal muscle-tendon/ligament complexes from other anatomical locations, as well as for single fibers and muscle strips.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>2-APB</td>
 <td>Tocris</td>
 <td>1224</td>
 <td>Blocker of a number of Ca2+ entry channels including SOC and TRP etc.</td>
 </tr>
 <tr>
 <td>SKF96365</td>
 <td>Sigma</td>
 <td>SKF-96365</td>
 <td>Blocker of a number of Ca2+ entry channels including SOC and receptor-mediated Ca2+ entry etc.</td>
 </tr>
 <tr>
 <td>BTP-2</td>
 <td>Millipore</td>
 <td>203890-5MG</td>
 <td>Relatively specific SOC blocker</td>
 </tr>
 <tr>
 <td>CPA</td>
 <td>Sigma</td>
 <td>C1530</td>
 <td>Reversible SERCA blocker</td>
 </tr>
 <tr>
 <td>caffeine</td>
 <td>Sigma</td>
 <td>C0750</td>
 <td>Fast action RyR agonist</td>
 </tr>
 <tr>
 <td>Radnoti Four Unit Tissue Organ Bath System</td>
 <td>Radnoti</td>
 <td>159920</td>
 <td></td>
 </tr>
 <tr>
 <td>Combination Tissue Support/Stimulating Electrode</td>
 <td>Radnoti</td>
 <td>160151</td>
 <td>Vertical Zig Zag Type with tissue support</td>
 </tr>
 <tr>
 <td>Quad Bridge Amp</td>
 <td>ADInstruments</td>
 <td>FE224</td>
 <td></td>
 </tr>
 <tr>
 <td>PowerLab/400</td>
 <td>ADInstruments</td>
 <td></td>
 <td>This product is no longer available. Choose other version of the data acquisition system.</td>
 </tr>
 <tr>
 <td>Force Transducers (5 mg - 25 g)</td>
 <td>ADInstruments</td>
 <td>MLT0201/RAD</td>
 <td></td>
 </tr>
 <tr>
 <td>Chart v4.02</td>
 <td>ADInstruments</td>
 <td></td>
 <td>LabChart 7.3 is the latest version of Chart software.</td>
 </tr>
 <tr>
 <td>S8800 Dual Pulse Digital Stimulator</td>
 <td>GRASS TECHNOLOGIES</td>
 <td></td>
 <td>This product is no longer available. S88X Dual Output Square Pulse Stimulator is a newer stimulator.</td>
 </tr>
 <tr>
 <td>RF Transformer Isolation Unit</td>
 <td>GRASS TECHNOLOGIES</td>
 <td>Model SIU5</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
 </table>

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.

Watch this Scientific Journal Video about Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles at JoVE.com

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.

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, ...

ex-vivo-assessment-contractility-fatigability-alternans-isolated

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JoVE Journal

Biology

23.5K ビュー.  UMDNJ-Robert Wood Johnson Medical School. 我々は、直接筋力、筋パワー、収縮動態との単離された骨格筋の疲労を測定する方法を説明します in vitroで電場刺激を用いたシステム。カルシウムについての貴重な情報 2 +ハンドリング性や筋肉の収縮機械が異なる刺激プロトコルを使用して取得できます。