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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+specific+force+and+power+production+of+muscle+fibers+from+myostatin-deficient+mice+are+associated+with+a+suppression+of+protein+degradation.">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><li>Gumucio, J. P., Davis, M. E., Bradley, J. R., Stafford, P. L., Schiffman, C. J., Lynch, E. B., Claflin, D. R., Bedi, A., Mendias, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rotator+cuff+tear+reduces+muscle+fiber+specific+force+production+and+induces+macrophage+accumulation+and+autophagy.">Rotator cuff tear reduces muscle fiber specific force production and induces macrophage accumulation and autophagy.</a> J Orthop Res. 30 (12), 1963-1970 (2012).</li><li>Mendias, C. L., Roche, S. M., Harning, J. A., Davis, M. E., Lynch, E. B., Sibilsky Enselman, E. r., Jacobson, J. A., Claflin, D. R., Calve, S., Bedi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+muscle+fiber+force+production+and+disrupted+myofibril+architecture+in+patients+with+chronic+rotator+cuff+tears.">Reduced muscle fiber force production and disrupted myofibril architecture in patients with chronic rotator cuff tears.</a> J Shoulder Elbow Surg. 1 (4), 111-119 (2015).</li><li>Moss, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcomere+length-tension+relations+of+frog+skinned+muscle+fibres+during+calcium+activation+at+short+lengths.">Sarcomere length-tension relations of frog skinned muscle fibres during calcium activation at short lengths.</a> J Physiol. 292, 177-192 (1979).</li><li>Chase, P. B., Kushmerick, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+pH+on+contraction+of+rabbit+fast+and+slow+skeletal+muscle+fibers.">Effects of pH on contraction of rabbit fast and slow skeletal muscle fibers.</a> Biophys J. 53, 935-946 (1988).</li><li>Moisescu, D. G., Thieleczek, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+and+strontium+concentration+changes+within+skinned+muscle+preparations+following+a+change+in+the+external+bathing+solution.">Calcium and strontium concentration changes within skinned muscle preparations following a change in the external bathing solution.</a> J Physiol. 275, 241-262 (1978).</li><li>Walker, S. M., Schrodt, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=I+Segment+lengths+and+thin+filament+periods+in+skeletal+muscle+fibers+of+the+Rhesus+monkey+and+the+human.">I Segment lengths and thin filament periods in skeletal muscle fibers of the Rhesus monkey and the human.</a> Anat Rec. 178, 63-81 (1974).</li><li>Gollapudi, S. K., Lin, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+determination+of+sarcomere+force-length+relationship+in+type-1+human+skeletal+muscle+fibers.">Experimental determination of sarcomere force-length relationship in type-1 human skeletal muscle fibers.</a> J Biomech. 42, 2011-2016 (2009).</li><li>Brenner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technique+for+stabilizing+the+striation+pattern+in+maximally+calcium-activated+skinned+rabbit+psoas+fibers.">Technique for stabilizing the striation pattern in maximally calcium-activated skinned rabbit psoas fibers.</a> Biophys J. 41 (1), 99-102 (1983).</li><li>Claflin, D. R., 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=Effects+of+high+and+low-velocity+resistance+training+on+the+contractile+properties+of+skeletal+muscle+fibers+from+young+and+older+humans.">Effects of high and low-velocity resistance training on the contractile properties of skeletal muscle fibers from young and older humans.</a> J Appl Physiol. 111, 1021-1030 (2011).</li><li>Lynch, G. S., Faulkner, J. A., Brooks, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+deficits+and+breakage+rates+after+single+lengthening+contractions+of+single+fast+fibers+from+unconditioned+and+conditioned+muscles+of+young+and+old+rats.">Force deficits and breakage rates after single lengthening contractions of single fast fibers from unconditioned and conditioned muscles of young and old rats.</a> Am J Physiol Cell Physiol. 295, C249-C256 (2008).</li><li>Frontera, W. R., Rodriguez Zayas, A., Rodriguez, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+of+human+muscle:+understanding+sarcopenia+at+the+single+muscle+cell+level.">Aging of human muscle: understanding sarcopenia at the single muscle cell level.</a> Phys Med Rehabil Clin N Am. 23, 201-207 (2012).</li><li>Malisoux, L., Francaux, M., Theisen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+do+single-fiber+studies+tell+us+about+exercise+training.">What do single-fiber studies tell us about exercise training.</a> Med Sci Sports Exerc. 39 (7), 1051-1060 (2007).</li><li>Widrick, J. L., Stelzer, J. E., Shoepe, T. C., Garner, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+properties+of+human+muscle+fibers+after+short-term+resistance+exercise+training.">Functional properties of human muscle fibers after short-term resistance exercise training.</a> Am J Physiol Regulatory Integrative Comp Physiol. 238, 408-416 (2002).</li><li>Trappe, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+spaceflight,+simulated+spaceflight+and+countermeasures+on+single+muscle+fiber+physiology.">Effects of spaceflight, simulated spaceflight and countermeasures on single muscle fiber physiology.</a> J Gravit Physiol. 9 (1), 323-326 (2002).</li><li>Malisoux, L., Jamart, C., Delplace, K., Nielens, H., Francaux, M., Thiesen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+long-term+muscle+paralysis+on+human+single+fiber+mechanics.">Effect of long-term muscle paralysis on human single fiber mechanics.</a> J Appl Physiol. 102, 340-449 (2006).</li><li>Krivickas, L. S., Walsh, R., Amato, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+muscle+fiber+contractile+properties+in+adults+with+muscular+dystrophy+treated+with+MYO-029.">Single muscle fiber contractile properties in adults with muscular dystrophy treated with MYO-029.</a> Muscle Nerve. 39, 3-9 (2009).</li><li>Russell, A. J., 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=Activation+of+fast+skeletal+muscle+troponin+as+a+potential+therapeutic+approach+for+treating+neuromuscular+diseases.">Activation of fast skeletal muscle troponin as a potential therapeutic approach for treating neuromuscular diseases.</a> Nature Medicine. 18 (3), 352-356 (2012).</li><li>Krivickas, L. S., Yang, J. I., Kim, S. K., Frontera, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+fiber+function+and+rate+of+disease+progression+in+amyotrophic+lateral+sclerosis.">Skeletal muscle fiber function and rate of disease progression in amyotrophic lateral sclerosis.</a> Muscle Nerve. 26, 636-643 (2002).</li><li>Mendias, C. L., Marcin, J. E., Calerdon, D. R., 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+EDL+and+soleus+muscles+of+myostatin-deficient+mice.">Contractile properties of EDL and soleus muscles of myostatin-deficient mice.</a> J Appl Physiol. 101, 898-905 (2006).</li><li>Lynch, G. S., Rafael, J. A., Chamberlain, J. S., 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=Contraction-induced+injury+to+single+permeabilized+muscle+fibers+from+mdx,+transgenic+mdx+and+control+mice.">Contraction-induced injury to single permeabilized muscle fibers from mdx, transgenic mdx and control mice.</a> Am J Physiol Cell Physiol. 279, 1290-1294 (2000).</li><li>Mizunoya, Q., Wakamatsu, J., Tatsumi, R., Ikeuchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+high-resolution+separation+of+rodent+myosin+heavy+chain+isoforms+in+a+mini-gel+electrophoresis+system.">Protocol for high-resolution separation of rodent myosin heavy chain isoforms in a mini-gel electrophoresis system.</a> Anal Biochem. 377, 111-113 (2008).</li><li>Hill, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heat+of+shortening+and+the+dynamic+constants+of+muscle.">The heat of shortening and the dynamic constants of muscle.</a> Proc R Soc Lond B Biol Sci. 126, 136-195 (1938).</li><li>Bottinelli, R., Canepari, M., Pellegrino, M. A., Reggiani, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-velocity+properties+of+human+skeletal+muscle+fibres:+myosin+heavy+chain+isoform+and+temperature+dependence.">Force-velocity properties of human skeletal muscle fibres: myosin heavy chain isoform and temperature dependence.</a> J Physiol. 495, 573-586 (1996).</li><li>Edman, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+velocity+of+unloaded+shortening+and+its+relation+to+sarcomere+length+and+isometric+force+in+vertebrate+muscle+fibres.">The velocity of unloaded shortening and its relation to sarcomere length and isometric force in vertebrate muscle fibres.</a> J Physiol. 291, 143-159 (1979).</li><li>Brenner, B., Eisenberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rate+of+force+generation+in+muscle:+Correlation+with+actomyosin+ATPase+activity+in+solution.">Rate of force generation in muscle: Correlation with actomyosin ATPase activity in solution.</a> PNAS. 83, 3542-3546 (1986).</li><li>Moss, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+calcium+on+the+maximum+velocity+of+shortening+in+skinned+skeletal+muscle+fibres+of+the+rabbit.">The effect of calcium on the maximum velocity of shortening in skinned skeletal muscle fibres of the rabbit.</a> J. Muscle Res. Cell Motil. 3, 295-311 (1982).</li><li>Pate, E., Wilson, G. J., Bhimani, M., Cooke, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+dependence+of+the+inhibitory+effects+of+orthovanadate+on+shortening+velocity+in+fast+skeletal+muscle.">Temperature dependence of the inhibitory effects of orthovanadate on shortening velocity in fast skeletal muscle.</a> Biophys J. 66, 1554-1562 (1994).</li><li>Ashley, C. C., Moisescu, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+changing+the+composition+of+the+bathing+solutions+upon+the+isometric+tension-pCa+relationship+in+bundles+of+crustacean+myofibrils.">Effect of changing the composition of the bathing solutions upon the isometric tension-pCa relationship in bundles of crustacean myofibrils.</a> J Physiol. 270, 627-652 (1977).</li><li>Godt, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-activated+tension+of+skinned+muscle+fibers+of+the+frog.+Dependence+on+magnesium+adenosine+triphosphate+concentration.">Calcium-activated tension of skinned muscle fibers of the frog. Dependence on magnesium adenosine triphosphate concentration.</a> J Gen Physiol. 63, 722-739 (1974).</li><li>Fabiato, A., Fabiato, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calculator+programs+for+computing+the+composition+of+the+solutions+containing+multiple+metals+and+ligands+used+for+experiments+in+skinned+skeletal+muscle+cells.">Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned skeletal muscle cells.</a> Journal de Physiologie (Paris). 75, 463-505 (1979).</li><li>Chase, P. B., Kushmerick, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+physiological+ADP+concentrations+on+contraction+of+single+skinned+fibers+from+rabbit+fast+and+slow+muscles).">Effect of physiological ADP concentrations on contraction of single skinned fibers from rabbit fast and slow muscles).</a> Am J Physiol. 268, C480-C489 (1995).</li><li>Godt, R. E., Maughan, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swelling+of+skinned+muscle+fibers+of+the+frog.">Swelling of skinned muscle fibers of the frog.</a> Biophysical Journal. 19, 103-116 (1977).</li><li>Kawai, M., Wray, J. S., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+lattice+spacing+change+on+cross-bridge+kinetics+in+chemically+skinned+rabbit+psoas+muscle+fibers.">The effect of lattice spacing change on cross-bridge kinetics in chemically skinned rabbit psoas muscle fibers.</a> Biophys J. 64, 187-196 (1993).</li><li>Millman, B. 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+filament+lattice+of+striated+muscle.">The filament lattice of striated muscle.</a> Physiol Rev. 78 (2), 359-391 (1998).</li><li>Edman, K. 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+mouse+single+muscle+fibers,+a+comparison+with+amphibian+muscle+fibers.">Contractile properties of mouse single muscle fibers, a comparison with amphibian muscle fibers.</a> J Exp Biol. 208, 1905-1913 (2005).</li><li>Phillips, S. K., Woledge, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+isometric+force,+maximum+power+and+isometric+heat+rate+as+a+function+of+sarcomere+length+in+mouse+skeletal+muscle.">A comparison of isometric force, maximum power and isometric heat rate as a function of sarcomere length in mouse skeletal muscle.</a> Pfl&#252;gers Archiv. 420, 578-583 (1992).</li><li>Stephenson, D. G., Williams, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+sarcomere+length+on+the+force-pCa+relation+in+fast+and+slow-twitch+skinned+muscle+fibres+from+the+rat.">Effects of sarcomere length on the force-pCa relation in fast and slow-twitch skinned muscle fibres from the rat.</a> J Physiol. 333, 637-653 (1982).</li></ol>

Production and free access to this article is sponsored by Aurora Scientific

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Polystyrene culture test tube with cap</td>
			<td>Fisher Scientific&#160;</td>
			<td>14-956-3D</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL screw cap micocentrifuge</td>
			<td>Fisher Scientific&#160;</td>
			<td>02-681-334</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL microcentrifuge caps with o-ring</td>
			<td>Fisher Scientific&#160;</td>
			<td>02-681-358</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge cryobox</td>
			<td>Fisher Scientific&#160;</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&#160;</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&#160;</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></td>
			<td></td>
			<td></td>
			<td>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>MgCl2&#8226;6H20</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>Na2H2ATP&#8226;0.56H2O</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>CaCO3</td>
			<td>Sigma-Aldrich</td>
			<td>C4830</td>
			<td></td>
		</tr>
		<tr>
			<td>NaN3</td>
			<td>Sigma-Aldrich</td>
			<td>S8032</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH (1N)</td>
			<td>Sigma-Aldrich</td>
			<td>35113</td>
			<td></td>
		</tr>
		<tr>
			<td>HCL (1N)</td>
			<td>Sigma-Aldrich</td>
			<td>318949</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2CrP&#8226;4H2O</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

Research

JoVE Journal

Bioengineering

25.0K vistas.  University of Michigan Medical School.  Análisis de las propiedades contráctiles de las fibras musculares, esqueléticas químicamente piel o permeabilizadas ofrece un poderoso medio de la cual evaluar la función muscular a nivel de la célula muscular individual. En este artículo describimos una técnica válida y fiable para preparar y probar permeabilized fibras musculares esqueléticas in vitro. 