Name: ex vivo assessment of contractility, fatigability and alternans in isolated skeletal muscles
Uploaded: 2012-11-01T12:00:00
Description: Watch this Scientific Journal Video about Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles Video at JoVE.com

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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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.

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Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles Video (Video) | JoVE

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, ...

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In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy Video (Video) | JoVE

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation Video (Video) | JoVE

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

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

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity Video (Video) | JoVE

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

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

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies Video (Video) | JoVE

Critical to the evaluation of potential  therapeutics for muscular disease are sensitive and reproducible physiological  assessments of muscle function. ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers Video (Video) | JoVE

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

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice Video (Video) | JoVE

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo Video (Video) | JoVE

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy Video (Video) | JoVE

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

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

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice Video (Video) | JoVE

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 ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo Video (Video) | JoVE

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...