This work was supported by the following funding sources: R01-AR063649, AG-020591, F31-AR035931.

<ol><li>Mendias, C. L., Kayupov, E., Bradley, J. R., Brooks, S. V., Claflin, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decreased+specific+force+and+power+production+of+muscle+fibers+from+myostatin-deficient+mice+are+associated+with+a+suppression+of+protein+degradation%5BTitle%5D)+AND+%22J+Appl+Physiol%22%5BJournal%5D)">Decreased specific force and power production of muscle fibers from myostatin-deficient mice are associated with a suppression of protein degradation.</a> J Appl Physiol. 111 (1), 185-191 (2011).</li>
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Assessments of the contractile properties of permeabilized single skeletal muscle fibers are used to investigate muscle function in a wide variety of contexts. Examples include studies that have evaluated the effects of aging12, exercise10,13,14, spaceflight15, injury2,3,16, drug treatments17,18, disease19 and genetic manipulation20,21 on fiber structure and function. Due to the ability to directly assess the contractile performance of myofibrils in their native configuration, this technique provides an attractive platform from which to form an understanding of myofibrillar function absent of potentially confounding effects that are present when neuromuscular signal transmission and excitation-induced calcium release are included in the system under study. Furthermore, functional testing of single fibers can be used to complement contractile protein identification results such as those obtained through immunohistochemistry or gel electrophoresis + western blot22.
One of the primary functions of skeletal muscle is to generate force. Consequently sFo, a measure of the intrinsic force generating capability of a contractile system, is of great interest to muscle physiologists. Reliable estimates of sFo require accurate measures of both fiber CSA and Fo. Since fibers are, in general, neither circular in cross-section, nor uniform in CSA along their length, great care should be taken when estimating CSA. To this end, measurements are made at several locations along the length of the fiber and, at each location, from two perspectives separated by 90&#176;. Reliable measures of Fo require attention to several details including accounting for passive force, adjusting sarcomere length to maximize overlap of thick and thin filaments, employing an activating solution with a calcium concentration that results in maximal activation, maintaining the desired experimental temperature, and maintaining optimal storage conditions (temperature and duration) of the fibers prior the day of the experiment.
While the steps outlined here describe the procedure for evaluating maximum isometric force, it is frequently desirable to evaluate other important functional qualities of skeletal muscle fibers. This can be achieved by extending the experimental protocol to include additional mechanical manipulations of the fiber. For example, measurement of the speed at which the fiber shortens against a series of different loads allows determination of the force-velocity relationship, from which force-power and velocity-power relationships can be computed10,23,24. Additionally, the speed of unloaded shortening can be determined by employing the &#8220;slack test&#8221;25, which consist of applying a series of slack-inducing shortening steps and measuring the time required by the fiber to remove the slack. Another kinetic parameter that is frequently reported is ktr, the rate constant for force redevelopment following a mechanical perturbation that temporarily detaches all crossbridges26. Finally, the relationship between calcium concentration and active force generation (the &#8220;force-pCa relationship&#8221;) is often of interest18 and can be determined by exposing the fiber to a series of solutions with calcium concentrations ranging from below the threshold for activating the contractile system to those sufficient to elicit maximum activation and therefore maximum force (Fo).
Though much of the mentioned equipment is needed for assessing single fiber contractility, other equipment is not absolutely necessary. The length-controller, for example, is essential for any experimental protocol that requires rapid or precise lengthening or shortening of the fiber, but is not absolutely necessary for evaluating maximum isometric force (though a zero-force level in the force record must still be identified by some means). The prisms that allow observation of the fiber from the side, while useful for assessing cross-sectional area, are not absolutely necessary when positioning the fiber within the experimental chamber. Furthermore, alternative means for exposing the fiber to the various experimental solutions could be employed, including devising a manually-operated system of chambers or a single chamber that allows for rapid filling and emptying of solutions. Finally, while sub-physiological experimental temperatures such as 15 &#176;C are commonly used to improve the reproducibility of mechanical measurements1,2,3,5,8,12,17,27, it is possible to generate valid data at other temperatures23,28 as long as the effects of temperature on solution properties (calcium concentration, pH, etc.) are taken into consideration.
The compositions of the testing solutions are among the most critical aspects of the permeabilized fiber techniques described here. Considerations regarding solution composition are complex and beyond the scope of this article. The solutions described in Step 5 of the protocol section are designed with an emphasis on rapid activation of the permeabilized fiber upon its transfer from pre-activating to activating solutions while maintaining a constant ionic strength, cationic composition, and osmolarity6,29. Other approaches to solution composition have been employed with notable success by other research groups and typically make use of published binding constants and computational tools27,30,31. The concentrations of calcium ions in the various activating solutions is particularly important in studies involving submaximal activation such as force-pCa evaluations. For experiments in which fibers are fully activated, such as those described here, the calcium concentration in the activating solution typically exceeds by a comfortable margin that required to achieve maximum force, making its precise knowledge less critical. Addition of creatine phosphate is important for buffering the intramyofibrillar ATP and ADP fluctuations that would otherwise be associated with contractile activity. Creatine kinase is required to catalyze the phosphate transfer from creatine phosphate to ADP. Under experimental conditions that result in high ATP turnover rates, including working at high temperatures or measuring high-speed shortening in fast fibers32, creatine kinase must be added to the solution to supplement the endogenous creatine kinase that remains bound to the fiber. For less demanding experimental conditions, the ATP regeneration system is less critical27.
Limitations of the permeabilized single fiber technique include the following. The data generated by these tests define the contractile properties of the specific myofibrillar unit that was attached to the experimental apparatus. Consequently, this captures only a small fraction of the entire multinucleated fiber from which the segment was obtained which, in turn, represents a small fraction of the total number of fibers within the muscle. Investigators should thus consider carefully the sampling required to support any conclusions drawn from the experiments. Additionally, evaluating the impact of an exercise training intervention on fiber function presumes that the fibers evaluated were indeed recruited during the training. Though the protocol attempts to mimic the natural intracellular milieu of the fiber, the sarcolemma permeabilization process is non-specific and necessarily allows soluble intracellular constituents to freely diffuse into the bathing solutions. A further consequence of the membrane permeability is a change in the osmotic balance evidenced by a swelling in fiber volume33. The fiber swelling increases the distance between actin and myosin filaments resulting in reduced calcium sensitivity of the myofilament system34,35, but can be reversed by the introduction of large, osmotically active compounds34. A final limitation to consider is the consequence of the technique used to attach fibers to the experimental apparatus. This invariably requires distorting the spatial relationship within the filament system at and near the attachment points, with attending functional deficits. Specifically, the regions of the fiber at and adjacent to the attachment points are functionally compromised and thereby contribute artifactual series elasticity to the measurement system.
In summary, we have described a means by which to assess the force-generating capacity of chemically permeabilized skeletal muscle fibers in vitro. Though the focus of this article has been on the assessment of maximum isometric force generating capacity of human skeletal muscle fibers, the experimental approach can be modified and extended to determine a variety of kinetic parameters and relationships across a range of species, mammalian or otherwise.

The primary function of skeletal muscle is to generate force. Muscle force is elicited in vivo through a complex sequence of events that includes motor nerve action potentials, neuromuscular transmission, muscle fiber action potentials, release of intracellular calcium, and activation of the system of regulatory and contractile proteins. Because force generation is the ultimate result of this sequence, a deficit in force could be caused by failure of one or more of the individual steps. A key attribute of the permeabilized fiber preparation is that it eliminates most of the steps required for force generation in vivo, with only the regulatory and contractile functions associated with the myofibrillar apparatus remaining. The investigator assumes control over the delivery of activating calcium and energy (ATP), and is rewarded with a simplified system that allows assessment of the isolated regulatory and contractile structures in their native configuration. Measurements of force using permeabilized skeletal muscle fibers are thus valuable when assessing alterations in muscle function observed in vivo. For example, we have used this technique to characterize the force generating capacity of fibers from myostatin deficient mice1 and to assess the cause of persistent muscle weakness exhibited following chronic rotator cuff tears2,3.
Modern permeabilized fiber methodology can be traced to early influential studies4,5 and is currently in use by a number of research groups. Though the techniques have been described in the literature, they have not yet been presented in video format. The goal of this article is to illustrate an updated, valid and reliable technique for measuring the maximum force generating capacity of single fibers from chemically permeabilized skeletal muscle samples. To accomplish this, an individual fiber segment (referred to herein as a &#8220;fiber&#8221;) is extracted from a pre-permeabilized bundle of fibers and placed in an experimental chamber containing a relaxing solution, the defining feature of which is a calcium concentration that is &#60;10 nM. The fiber is then attached at one end to a force-transducer and at the other end to a length-controller. With the fiber held at an optimal sarcomere length, it is transferred to an activating solution that has a calcium concentration sufficient to elicit maximum activation and thereby maximum isometric contraction force. Force data are acquired, stored and analyzed using a personal computer.

<table><tbody><tr><td>Polystyrene culture test tube with cap</td><td>Fisher Scientific&amp;nbsp;</td><td>14-956-3D</td><td></td></tr><tr><td>0.5 ml screw cap microcentrifuge</td><td>Fisher Scientific&amp;nbsp;</td><td>02-681-334</td><td></td></tr><tr><td>0.5 ml microcentrifuge caps with o-ring</td><td>Fisher Scientific&amp;nbsp;</td><td>02-681-358</td><td></td></tr><tr><td>Microcentrifuge cryobox</td><td>Fisher Scientific&amp;nbsp;</td><td>5055-5005</td><td></td></tr><tr><td>pH meter</td><td>Mettler-Toledo</td><td>FE20</td><td></td></tr><tr><td>Petri dish</td><td>Fisher Scientific&amp;nbsp;</td><td>08-757-11YZ</td><td></td></tr><tr><td>Nonsterile-suture 10-0 monofilament</td><td>Ashaway Line Twine</td><td>S30002</td><td></td></tr><tr><td>Insect pins</td><td>Fine Science Tools</td><td>26002-10</td><td></td></tr><tr><td>Forceps - Dumont #5</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Microdissecting scissors</td><td>Fine Science Tools</td><td>15000-08</td><td></td></tr><tr><td>Stereo microscope</td><td>Leica Microsystems</td><td>MZ8</td><td></td></tr><tr><td>Micrometer drives</td><td>Parker Hannifin</td><td>3936M</td><td></td></tr><tr><td>Thermometer</td><td>Physitemp</td><td>BAT-12</td><td></td></tr><tr><td>Water bath circulator&amp;nbsp;</td><td>Neslab Instruments</td><td>RTE-111</td><td></td></tr><tr><td>Temperature controller</td><td>Aplha Omega Instruments</td><td>Series 800</td><td></td></tr><tr><td>LabVIEW software</td><td>National Instruments</td><td>-</td><td></td></tr><tr><td>Computer</td><td>Varied</td><td>-</td><td></td></tr><tr><td>Chamber system</td><td>Aurora Scientific</td><td>802D</td><td></td></tr><tr><td>Length-controller</td><td>Aurora Scientific</td><td>312C</td><td></td></tr><tr><td>Force-transducer</td><td>Aurora Scientific</td><td>403A</td><td></td></tr><tr><td colspan="4">Reagents</td></tr><tr><td>K-proprionate</td><td>TCI America</td><td>P0510</td><td></td></tr><tr><td>Imadizole</td><td>Sigma-Aldrich</td><td>I0125</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;6H&lt;sub&gt;2&lt;/sub&gt;0</td><td>Sigma-Aldrich</td><td>M2670</td><td></td></tr><tr><td>Brij 58</td><td>Sigma-Aldrich</td><td>P5884</td><td></td></tr><tr><td>EGTA (acid)</td><td>Sigma-Aldrich</td><td>E0396</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;H&lt;sub&gt;2&lt;/sub&gt;ATP&amp;bull;0.56H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>A7699</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G6279</td><td></td></tr><tr><td>HEPES (acid)</td><td>Sigma-Aldrich</td><td>H7523</td><td></td></tr><tr><td>MgO</td><td>Sigma-Aldrich</td><td>529699</td><td></td></tr><tr><td>HDTA (acid)</td><td>TCI America</td><td>D2019</td><td></td></tr><tr><td>CaCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C4830</td><td></td></tr><tr><td>NaN&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S8032</td><td></td></tr><tr><td>KOH (1 N)</td><td>Sigma-Aldrich</td><td>35113</td><td></td></tr><tr><td>HCL (1 N)</td><td>Sigma-Aldrich</td><td>318949</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;CrP&amp;bull;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>P7936</td><td></td></tr><tr><td>pH 10 standard</td><td>Fisher Scientific</td><td>SB115</td><td></td></tr><tr><td>pH 7 standard</td><td>Fisher Scientific</td><td>SB107</td><td></td></tr></tbody></table>

measurement of maximum isometric force generated by permeabilized skeletal muscle fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Healthy, chemically permeabilized single fibers should appear uniform in shape and have consistent striation spacing when viewed under high magnification. Fibers that are inflexible when manipulated with the forceps or have obvious structural damage should be discarded.
High magnification digital images taken during step 15 are analyzed for 5 paired diameter measurements along the midsection of the fiber. Fiber CSA is estimated assuming an elliptical cross section and averaging 5 individual CSA measurements as depicted in Figure 7A. Figure 7B also serves to illustrate how fiber dimensions in one view can be significantly different compared with paired dimensions in the other view (i.e., cross-sections are not, in general, round).
Representative force traces from human slow and fast fibers are shown in Figures 8A and 8B, respectively. Voltage output of the force-transducer is acquired during a test and converted to force (mN) using data acquisition and analysis software (LabVIEW). Figure 9 illustrates the approach used to assess maximum active force (Fo), which is calculated by subtracting the force required to maintain the fiber at optimal sarcomere length while in a relaxed state (passive force, FP), from the greatest isometric force developed during maximal fiber activation (total force, FT). Since the output of the force-transducer that corresponds to zero force is, in general, different for each of the different bathing chambers, we briefly slacken the fiber in both the pre-activating and activating solutions to capture the zero-force level in the experimental record. Normalization of maximum active force by fiber CSA is used to generate the more informative value of specific force (sFo). Because it takes into consideration the CSA of the fiber, sFo provides a measure of the intrinsic force generating capacity of the fiber&#8217;s contractile apparatus, thereby allowing functional comparisons between fibers of disparate sizes. It should, however, be noted that CSA measurements are not able to distinguish the proportion of the fiber occupied by contractile filaments versus the proportion occupied by other subcellular structures.
Typical characteristics of healthy, adult fibers from Claflin et al. 201110 for human, Mendias et al. 20111 for mouse and Gumucio et al. 20122 for rat are detailed in Table 3. All data presented in Table 3 were generated using the techniques described in this article.
<table border="0" cellpadding="0" cellspacing="0" style="text-align:center">
	<tbody>
		<tr>
			<td rowspan="3" style="border:0px #fff solid"></td>
			<td colspan="4">Human 
			(vastus lateralis)</td>
			<td>Mouse 
			(EDL)</td>
			<td nowrap="nowrap">Rat 
			(infraspinatus)</td>
		</tr>
		<tr>
			<td colspan="2">Male</td>
			<td colspan="2">Female</td>
			<td>Male</td>
			<td>Male</td>
		</tr>
		<tr>
			<td>Type 1</td>
			<td>Type 2a</td>
			<td>Type 1</td>
			<td>Type 2a</td>
			<td>(not typed)</td>
			<td>(not typed)</td>
		</tr>
		<tr>
			<td>CSA (&#956;m2)</td>
			<td>4880 - 6900</td>
			<td>5270 - 8380</td>
			<td>3870 - 5470</td>
			<td>4010 - 5610</td>
			<td>1850 - 3080</td>
			<td>5290 - 8010</td>
		</tr>
		<tr>
			<td>Fo (mN)</td>
			<td>0.79 - 1.17</td>
			<td>1.02 - 1.54</td>
			<td>0.64 - 0.97</td>
			<td>0.71 - 1.07</td>
			<td>0.14 - 0.25</td>
			<td>0.55 - 0.97</td>
		</tr>
		<tr>
			<td>sFo (kPa)</td>
			<td>142 - 182</td>
			<td>165 - 210</td>
			<td>156 - 193</td>
			<td>172 - 214</td>
			<td>67 - 94</td>
			<td>102 - 131</td>
		</tr>
		<tr>
			<td>n</td>
			<td>129</td>
			<td>160</td>
			<td>149</td>
			<td>207</td>
			<td>37</td>
			<td>94</td>
		</tr>
	</tbody>
</table>

Table 3. Typical characteristics of healthy, adult fibers from human vastus lateralis10, mouse extensor digitorum longus1 and rat infraspinatus2 muscles. Optimal sarcomere lengths were set at 2.7 &#181;m for human fibers7,8 and 2.5 &#181;m for both mouse (36,37) and rat fibers (38). Experimental Lf ranges (25th and 75th quartiles) were 1.39-1.73 mm, 1.17-1.53 mm and 1.32-1.59 mm for human, mouse and rat respectively. Ranges shown indicate the 25th and 75th quartiles and n is the number of fibers tested.
The most common problems experienced during testing include a suture loop slip, which results in a force response with a &#8220;catch&#8221; such as that illustrated in Figure 10A, and a partial or full thickness tear of the fiber, which results in a force response that returns abruptly toward or to (break) zero while the fiber is still immersed in activating solution (Figure 10B). If a slip, tear or break occurs during an experiment, the fiber should be discarded and the data excluded, though maintaining a record of fiber failures can also be informative11. Another negative outcome that may be encountered is the premature activation of the fiber while in the pre-activating solution (Figure 10C). Partial activation in the pre-activating solution suggests significant cross-well contamination (i.e., an unintentional increase in calcium concentration in the pre-activating well). In this instance, all baths should be aspirated and rinsed well with deionized water. Drying the dividing surfaces between the chambers is also recommended as moisture or condensation in these areas may lead to wicking of solution between baths. The decision to include or exclude data will ultimately depend on the experimental focus and should thus be considered when designing the study.
<img alt="Figure 1" src="/files/ftp_upload/52695/52695fig1.jpg" /> 
Figure 1: Suture loop (10-0 monofilament nylon suture).
<img alt="Figure 2" src="/files/ftp_upload/52695/52695fig2.jpg" /> 
Figure 2: Bundle dissection. Forceps are in left hand, microdissection scissors are in right hand. Red line indicates the favorable orientation of the wrist and scissors with the longitudinal axes of the fibers.
<img alt="Figure 3" src="/files/ftp_upload/52695/52695fig3.jpg" /> 
Figure 3: (A) Testing apparatus with labeled components. (a) Experimental chambers with transparent bottoms. (b) Length-controller. (c) Force-transducer. (d) Light source. (e) Length-controller x-y-z micrometer drive with digital display. (f) Stage micrometer drive with digital display. (g) Force-transducer x-y-z micrometer drive. (h) Platform for calibrated laser-diffraction target screen. (i) Vibration isolation table. (B) Close-up view of the experimental chambers. (j) Stainless steel attachment surface extending from the length-controller. (k) Stainless steel attachment surface extending from the force-transducer. (l) Side-view prism. (m) Housing for thermoelectric cooling modules. (n) Thermocouple for reporting chamber temperature.
<img alt="Figure 4" src="/files/ftp_upload/52695/52695fig4.jpg" /> 
Figure 4: Modified 100 &#181;l pipet tip used to transfer fiber from dissection dish to experimental chamber.
<img alt="Figure 5" src="/files/ftp_upload/52695/52695fig5.jpg" /> 
Figure 5: Mounting single fiber onto experimental apparatus. (A) Prepared suture loops threaded onto stainless steel attachment surfaces. (B) Fiber transferred to experimental chamber. (C) Fiber anchored to stainless steel attachment surfaces by first pair of suture loops with excess suture removed. (D) Second pair of suture loops threaded over the top of first suture loops and tied in place.
<img alt="Figure 6" src="/files/ftp_upload/52695/52695fig6.jpg" /> 
Figure 6: Sarcomere length is assessed by the projection of a laser interference pattern onto a calibrated target screen. (a) Laser source. (b) Mirror. (c) Target screen. (d) Laser interference pattern.
<img alt="Figure 7A" src="/files/ftp_upload/52695/52695fig7A.jpg" />
<img alt="Figure 7B" src="/files/ftp_upload/52695/52695fig7B.jpg" /> 
Figure 7: (A) Determination of fiber cross-sectional area at optimal sarcomere length (human = 2.7 &#181;m). Assuming an elliptical cross-section, CSA is calculated for each of five locations along the fiber midsection and the mean of the five individual measurements is reported as fiber CSA. 2a represents top view diameter and is one axis of the ellipse, 2b represents side view diameter and is the other axis of the ellipse. (B) Representative fiber images illustrating each of the five corresponding diameter measurements taken in both the top and side view.
<img alt="Figure 8" src="/files/ftp_upload/52695/52695fig8.jpg" /> 
Figure 8: Representative force traces from healthy human vastus lateralis muscle fibers. (A) Type 1 fiber (CSA: 5710 &#181;m2, Fo: 0.89 mN and sFo: 156 kPa). (B) Type 2a fiber (CSA: 9510 &#181;m2, Fo: 1.66 mN and sFo: 174 kPa). Fiber myosin heavy chain type was determined through the use of electrophoretic separation and silver-staining techniques22.
<img alt="Figure 9" src="/files/ftp_upload/52695/52695fig9.jpg" /> 
Figure 9: Calculation of maximum active force (Fo). (a) Expanded view of fiber force response during slack-inducing movement of length-controller initiated in pre-activating solution. FP is the force required to maintain a sarcomere length of 2.7 &#181;m with the fiber at rest. (b) Expanded view of length-controller slack-inducing movement. Note that Fo = FT&#8211; FP.
<img alt="Figure 10" src="/files/ftp_upload/52695/52695fig10.jpg" /> 
Figure 10: (A) Suture loop slip, evidenced by a &#8220;catch&#8221; in force trace during the rise of force. Check to be sure loops are secure before activating the fiber. (B) Fiber break during activation. May be due to poor fiber integrity or aggressive fiber treatment during suture loop placement. (C) Premature partial fiber activation due to contamination of pre-activation chamber with Ca2+.

Watch this Scientific Journal Video about Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers at JoVE.com

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

measurement-maximum-isometric-force-generated-permeabilized-skeletal

Nanyang Technological University

Research

JoVE Journal

Bioengineering

25.4K Views.  University of Michigan Medical School.  Analyse van de contractiele eigenschappen van chemisch huid of doorlaatbaar gemaakt, skeletspier vezels een krachtig middel om spierfunctie beoordeeld op het niveau van de interne spiercel. In dit artikel schetsen we een valide en betrouwbare techniek bereid en getest doorlaatbaar skeletspier vezels in vitro. 

Video: Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

Medicine

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Neuroscience

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

Biology

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, ...

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. ...

Maximum Isometric Tetanic Force Measurement of the Tibialis Anterior Muscle in the Rat

Traumatic nerve injuries result in substantial functional loss and segmental nerve defects often necessitate the use of autologous interposition nerve grafts. Due to their limited availability and associated donor side morbidity, many studies in the field of nerve regeneration focus on alternative techniques to bridge a segmental nerve gap. In order to investigate the outcomes of surgical or pharmacological experimental treatment options, the rat sciatic nerve model is often used as a bioassay. There are a variety of outcome measurements used in rat models to determine the extent of nerve regeneration. The maximum output force of the target muscle remains the most relevant outcome for clinical translation of experimental therapies. Isometric force measurement of tetanic muscle contraction has previously been described as a reproducible and valid technique for evaluating motor recovery after nerve injury or repair in both rat and rabbit models. In this video, we will provide a step-by-step instruction of this invaluable procedure for assessment of functional recovery of the tibialis anterior muscle in a rat sciatic nerve defect model using optimized parameters. We will describe the necessary pre-surgical preparations in addition to the surgical approach and dissection of the common peroneal nerve and tibialis anterior muscle tendon. The isometric tetanic force measurement technique will be detailed. Determining the optimal muscle length and stimulus pulse frequency is explained and measuring the maximum tetanic muscle contraction is demonstrated.

Evaluation of motor recovery remains the benchmark outcome measure in experimental peripheral nerve studies. The isometric tetanic force measurement of the tibialis anterior muscle in the rat is an invaluable tool to assess functional outcomes after reconstruction of sciatic nerve defects. The methods and nuances are detailed in this article.

Traumatic nerve injuries result in substantial functional loss and segmental nerve defects often necessitate the use of autologous interposition nerve ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...

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 ...

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 ...

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

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