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Lateral Force Transmission Across Costameres in Skeletal Muscle

Bloch, Robert J.; Gonzalez-Serratos, Hugo

Exercise and Sport Sciences Reviews: April 2003 - Volume 31 - Issue 2 - p 73-78
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BLOCH, R. J., and H. GONZALEZ-SERRATOS. Lateral force transmission across costameres in skeletal muscle. Exerc. Sport Sci. Rev., Vol. 31, No. 2, pp. 73–78, 2003. In skeletal muscle, contractile force can be transmitted laterally between the z-disks and M-lines of neighboring myofibrils, across the sarcolemma, and through the extracellular matrix to the tendon. Here we examine the data that support this model and the sarcolemmal properties and structures, termed “costameres,” that are consistent with it.

Department of Physiology, University of Maryland School of Medicine, Baltimore

Accepted for publication: January 22, 2003.

Address for correspondence: Robert J. Bloch, Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201 (E-mail: rbloch@umaryland.edu).

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INTRODUCTION

The sarcolemma of skeletal muscle is subjected to considerable stress during the contractile cycle. One of the mechanisms that protects the sarcolemma against contractile damage is the formation of regular connections with the underlying contractile apparatus. These connections are made at structures called costameres.

Costameres were first recognized as rib-like structures (“costa,” Latin for “rib”) overlying the z-lines of nearby myofibrils (7). The term has since been used to describe similar structures that lie at the sarcolemma over M-lines and, in some muscles, parallel to the long axis of the myofiber (Fig. 1). These regular connections allow the sarcolemma to move in unison with the z- and M-lines of nearby myofibrils, with which it is aligned (Fig. 2B). As a result, the intercostameric regions of the sarcolemma may bulge out slightly (Fig. 2C). These bulges, or “festoons” (13), are indicative of the presence of very firm connections between the sarcolemma and the underlying contractile apparatus (13).

Figure 1

Figure 1

Figure 2

Figure 2

The presence of costameres and the formation of festoons at the sarcolemma have important physiological consequences. As mentioned above, they ensure that the displacements and distortions of the sarcolemma of contracting muscle are small and periodic. In addition, they contribute to a pathway by which the force of contraction can be transmitted laterally, from myofibril to myofibril, across the sarcolemma to the extracellular matrix, and ultimately through the extracellular matrix to the tendons. Here we review the evidence for lateral force transmission and the features of costameres that are consistent with it.

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PATHWAYS OF FORCE TRANSDUCTION

The sliding-filament model of muscle contraction states that force is generated when the contractile elements are activated, and the thick myosin filaments pull on the thin actin filaments. Once the external load is overcome, each sarcomere shortens (Fig. 2). Shortening is seen in the reduced size of the I bands (where actin is not covered by myosin) and in the reduced distance between the z-disks, which are composed of a paracrystalline array of α-actinin that anchors the actin filaments. As all sarcomeres and all myofibrils in a muscle fiber are activated simultaneously during contraction, the shrinking of the I bands and the moving together of neighboring z-disks (which occurs during isotonic or semi-isometric contractions) make it appear as if the force generated by the actomyosin interactions within each sarcomere is being transmitted longitudinally to the next sarcomere in the myofibril (Fig. 2B, small arrows), and so on until the myofibril terminates in the vicinity of the myotendinous junction. There, the contractile elements are linked to the junctional membrane and, through it, to the tendon. This “longitudinal pathway” of force transduction is commonly accepted, despite the fact that it has never been rigorously tested against alternative models.

One alternative model proposes that contractile force is transmitted laterally through a series of radially oriented elastic elements, from the z-disk of one myofibril to the z-disk of a parallel myofibril, and ultimately from the most superficial myofibril to the sarcolemma (Fig. 2B, large arrows). Transduction then occurs through the sarcolemma, which, by definition, includes not only the plasma membrane but also the closely apposed intracellular and cytoskeletal structures, as well as the extracellular matrix (14). Together, these structures form an elastic element parallel to the long axis of the fiber that also ends at the myotendinous junction.

Structural studies of skeletal muscle fibers are consistent with both models, as the links required for each are present at the appropriate sites in the myoplasm. As mentioned above, the longitudinal transmission of force could in principle occur via the serially arranged z-disks within a myofibril. Each myofibril terminates at the sarcolemma at the myotendinous junction, which is highly infolded to create additional sites of attachment for both the contractile elements of the last half-sarcomere and the extracellular matrix that anchors the collagen fibrils of the tendon. The cytoplasmic surface of the sarcolemma at the myotendinous junction is also highly enriched in cytoskeletal proteins that help to anchor the actin filaments of the last half-sarcomere to the membrane. Consistent with the longitudinal model of force transmission, the myotendinous junction is a common site of damage when myofibers are overloaded.

Structures are also present within and between myofibrils, and between superficial myofibrils and the sarcolemma, to provide a mechanical pathway for lateral transmission of force. The paracrystalline array of α-actinin at each z-disk is of course highly stable, and it is reinforced by the presence of several proteins that interact either directly or indirectly with the α-actinin matrix. Likewise, the M-lines of each sarcomere are highly crosslinked and stabilized by “M bridges” and associated structures. Structural elements, the nature of which remains controversial, align individual myofibrils with their neighbors, as z-disks and M-lines can be traced across a myofiber with only slight displacements. Actin filaments and intermediate filaments, including desmin- and cytokeratin-based filaments, are the most likely candidates to link the superficial myofibrils to the cytoplasmic surface of the sarcolemma (see below). Costameres, the sites at the sarcolemma where these filaments attach (13), contain large membrane-cytoskeletal complexes that are thought to reinforce the membrane and its links to the contractile elements and the extracellular matrix. Lateral force transmission is consistent with the observations that the force of contraction increases as myofibers increase in radius, not in length, and that myofibers in a fascicle move in unison, even when only one is activated.

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Distinguishing Between the Longitudinal and Lateral Pathways

Although structural studies cannot distinguish between these models, physiological studies of skeletal muscle have provided evidence for the lateral pathway. The results fall into two categories: experiments that tested the ability of the sarcolemma to transmit the force of contraction (13), and experiments that we have reevaluated to determine the relationship between contractile force and the cross-sectional dimensions of the fiber (4,9). Both approaches used frog twitch muscle and were performed with single muscle fibers or small fiber bundles. The former experiments (13) used a preparation in which a small bundle of muscle fibers was crushed to induce “retraction clots” of myofibrillar materials. These clots separated from the sarcolemma within each fiber, leaving sarcolemmal tubes that were clear of contractile proteins, as judged by light microscopy. The myofibers were stimulated electrically to develop maximal tetanic force, which was measured by transducers attached to one of the tendons. Comparison of the contractile force before and after the clot was induced showed only a 20–30% decrease following formation of the clot. As the sarcolemma was the only structure that was clearly retained between the clot and the myotendinous junction, this suggests that ∼75% of the force of contraction was carried from the actively contracting portion of the myofiber through the sarcolemma to the junction via a lateral pathway. Thus, no more than 20% to 30% of the force of contraction in this preparation is transmitted longitudinally.

In a second experimental paradigm (13), a single muscle fiber was isolated along its distal third but remained attached to nearby cut fibers in a small bundle. The nearby fibers, which formed a “splint,” were firmly attached to the dish; the tendon, which was shared by the splint and the partially isolated fiber, was attached to a force transducer. The other, loose end of the partially isolated fiber was subjected to direct tetanic stimulation and the force transmitted via the splinted region to the tendon was measured. More than 80% of the force was transmitted through the splint. This, too, suggests that most of the force of contraction can be transmitted laterally from one muscle fiber to its neighbors, and ultimately to the tendon.

The relationship of the force of contraction to fiber dimensions can also help to distinguish between longitudinal and lateral pathways of force transmission. We have reevaluated two sets of experimental data (4,9) that measured the contractile force generated by individual muscle fibers to learn if force is a linear or square function of the radius of the fiber, if fibers are modeled as cylinders, or of the major axis, if they are modeled as ellipses. In either case, if we assume a unit volume of constant length, the longitudinal pathway predicts that the force generated by single muscle fibers is a function of the cross-sectional area of the muscle fiber, and thus a square function of the radius or major axis, whereas a lateral pathway predicts that force is a function of the perimeter of the muscle fiber, and thus a linear function of the radius or major axis. Two groups did careful measurements of the force generated by myofibers of different sizes (4,9). When we recalculated their data to determine the relationship of force to radius or major axis, the results from both groups fell on a line with a slope of 1.18 N·cm−1 (Fig. 3). They clearly did not vary as a square function. We obtained similar results if we assumed the cross-sections of the myofibers were triangular (not shown).

Figure 3

Figure 3

Thus, most of the force of contraction appears to be transmitted laterally, across the sarcolemma, rather than longitudinally, through the sarcoplasm. These data are not definitive, however. For example, sarcolemmal tubes may retain structural elements, such as microtubules or intermediate filaments, that could transmit force longitudinally. Similarly, the linear dependence of force on fiber radius or major axis does not point specifically to a role for the sarcolemma in force transduction.

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HOW STRONG IS THE SARCOLEMMA AND ITS CONNECTIONS TO THE UNDERLYING CONTRACTILE APPARATUS?

The biomechanical properties of the sarcolemma support the notion that the sarcolemma and its connections to the contractile apparatus are able to withstand the forces of contraction in healthy skeletal muscle. Studies of the sarcolemma in actively contracting muscle have been conducted with the clotted fiber preparation, described above. Qualitatively, the sarcolemmal tube distal to the clot was strong enough to transmit all of the contractile force generated by the damaged fiber, as it did not tear when the fiber was stimulated. Measurements of the breaking stress of the sarcolemma yielded values of 26.7 N·cm−2 (4), or ∼18% less than the tetanic tension of 36 N·cm−2 (0.36 MPa; 1 MPa = 102 N·cm−2 (4,9,14)). However, the former value was calculated based on the cross-sectional area of the entire sarcolemmal tube, including its presumably empty interior, rather than on the sarcolemma itself. Correcting for this, the value is ∼12 × 103 N·cm−1 (2), close to the value of 5 × 103 N·cm−1 determined by other investigators (14). These values, which are comparable with the failure strength of muscle tendons, are likely to be overestimates, but they nevertheless indicate that the sarcolemma can withstand forces of contraction several-hundred–fold higher than those generated physiologically.

Connections between the sarcolemma and the underlying contractile apparatus have been studied in two ways. In one experiment, the sarcolemma was physically removed from half of the fiber length and the effects of stretching the myofiber were assessed (8). The results suggest that the resting sarcolemma can limit the extent to which sarcomeres lengthen, and indicated that 20% of the passive elastic modulus of the muscle could be attributed to attachments between the myofibrils and the sarcolemma. In a second experiment (10), a large suction pipet was placed on the (resting) sarcolemma and the distensibility of the membrane and attached contractile apparatus was studied at increasing negative pressures. The tension exerted by the membrane decreased at pressures above those that broke its connections between the contractile apparatus. The difference in tension exerted by the intact and disconnected preparations was ∼2 × 10−4 N·cm−1, which we ascribe to the links between the costameres and the contractile apparatus. The difference in stress exerted by the intact and disconnected preparations, adjusted for the fractional membrane area occupied by the costameres (∼35%), is ∼60 N·cm−1 (2), or ∼1.7 times higher than the maximal tetanic tension. These results are consistent with the existence of bridges between costameres at the sarcolemma and the contractile apparatus that are strong enough to transmit the forces normally exerted during the contractile cycle.

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STRUCTURAL ELEMENTS THAT MAY MEDIATE LATERAL FORCE TRANSMISSION AT COSTAMERES

Four sets of structural elements mediate lateral force transmission from the contractile apparatus through the superficial myofibrils and the sarcolemma, and via the extracellular matrix to the tendon: structures that connect myofibrils to one another at the level of M and z-lines; microfilaments and intermediate filaments that link myofibrils to the membrane; costameres, which anchor those filaments, span the sarcolemmal membrane, and interact with the extracellular matrix; and the extracellular matrix, which surrounds the myofiber from tendon to tendon.

The element that is most likely to withstand high contractile forces is the extracellular matrix. This structure, composed of the basal lamina and the reticular lamina (12) (see Fig. 4) contains collagen fibrils that are cross-linked for stability, and that are also bound to proteoglycans and noncollagenous glycoproteins, such as fibronectin. The filaments of collagen in the reticular lamina (12) are not easily stretched or broken and so probably form the most rigid elements in the matrix. They are also the elements most likely to link one myofiber to its neighbors. The basal lamina, to which the collagen fibrils are anchored, closely apposes the muscle plasma membrane and is composed of several proteins that interact with each other in different ways to form a stable, cross-linked structure. Laminin may be the most important of these basal laminar proteins, at least in mammalian muscle, as the absence of its α2 subunit causes congenital muscular dystrophy in humans and a similar dystrophy in mice.

Figure 4

Figure 4

The basal lamina is linked to the muscle plasma membrane by at least two kinds of receptors, dystroglycan and integrins (Fig. 4). Integrins of the vitronectin receptor family, containing the αv subunit, are present at costameres. In addition, a splice variant of α7β1-integrin, α7Cβ1D, selectively binds to laminin in the muscle basal lamina (3). Increasing the expression of this integrin can stabilize dystrophic muscle against contractile damage, whereas decreasing its expression may contribute to the pathology seen in congenital laminin deficiencies, as mentioned above. Dystroglycan also serves as a laminin receptor (5). As mutations that reduce dystroglycan’s binding activity also result in muscular dystrophy, laminin-dystroglycan interactions probably contribute significantly to the lateral pathway of force transmission.

Dystroglycan spans the sarcolemmal plasma membrane and binds to peripheral membrane proteins in the cytoplasm to form the next link in the lateral pathway of force transmission (Fig. 4). The cytoplasmic portion of dystroglycan binds a variety of proteins, including dystrophin, the structural protein that is missing in young boys with Duchenne muscular dystrophy. Defects in the linkage of dystroglycan to dystrophin probably underlie most of the pathology seen in Duchenne muscular dystrophy patients, as the region of dystrophin that is invariably affected contains the binding site for dystroglycan (5). Defects in dystrophin’s ability to bind other sarcolemmal proteins, including dystrobrevin and syntrophin, have little or no affect on the health of skeletal muscle. Few studies of contractile force in mice missing these proteins have been done, however.

Dystroglycan may also associate with cytoplasmic elements through the sarcoglycan complex (Fig. 4), a group of transmembrane proteins that bind to dystroglycan. To date, only one cytoplasmic ligand for the sarcoglycans has been found: filamin 2, a structural protein that binds actin and other cytoplasmic proteins. Thus, the lateral pathway for force transmission is likely to depend heavily on dystroglycan and its ability to link laminin and the extracellular matrix to dystrophin and other structural proteins at costameres.

The number of cytoplasmic proteins at costameres is large and still growing. In addition to dystrophin, dystroglycan, and their associated proteins, which are enriched at costameres (2), costameres have at least two other membrane-cytoskeletal complexes, one containing vinculin and another containing spectrin and ankyrin (Fig. 4).

Vinculin, the first protein to be localized at costameres (7), is the quintessential protein of focal adhesions, where it colocalizes with paxillin, talin, α-actinin, integrins, and signaling proteins. Surprisingly little has been learned about the role of any of these proteins at costameres, however. Studies of the pathophysiology of the dystrophin complex have advanced rapidly through the use of “knock-out” mice, but mice missing vinculin die in utero.

Like dystrophin, to which it is related structurally, spectrin also forms a network of filaments at the muscle plasma membrane. These filaments are anchored to the membrane indirectly, through an intermediate protein, called ankyrin, which in turn binds to integral proteins, including the Na+K+ATPase, and probably the Na+ channel, to anchor them to costameres (2). The spectrin at costameres has some unusual properties, but it may not be essential for muscle integrity, as mice lacking the β subunit of muscle spectrin are not myopathic. The dystrophin, spectrin, and vinculin complexes may interact with each other at the membrane. Such interactions are indicated by the observation that the voltage-gated Na channel can bind both to dystrophin via syntrophin, and to spectrin via ankyrin. Consistent with this notion, dystrophic (mdx) mouse muscle shows extensive but coordinated rearrangement of all its membrane-cytoskeletal proteins, including proteins of the vinculin, spectrin, and dystrophin complexes (15). This suggests that these proteins are linked to each other either directly or indirectly, even in the absence of dystrophin.

The sarcolemma is linked to the contractile apparatus by microfilaments and intermediate filaments. Microfilaments bind to dystrophin in vitro and are linked in situ to the dystrophin complex at costameres, especially near z-lines (11). Intermediate filaments composed of desmin and synemin also link z-lines to costameres, and may well be anchored at the membrane through synemin, which copolymerizes with desmin and binds vinculin (1). These structures are not present in significant amounts at costameres that overlie M-lines or that lie in longitudinal arrays at the sarcolemma, however, suggesting that other structures must be involved.

We have recently identified a second intermediate filament system at those sites, as well as at the level of z-lines, and that can persist even in the absence of desmin (6). Cytokeratin filaments, too, anchor peripheral membrane proteins at costameres. Additional studies of the properties of the sarcolemma in muscles lacking desmin, synemin, cytokeratin, and, if possible, cortical actin, should reveal the relative contributions of each of these filamentous structures to the connections between neighboring myofibrils and to lateral force transmission.

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CONCLUSION

The sarcolemma of skeletal muscle is strong enough to transmit the force of contraction, but the evidence that force transmission follows a lateral pathway across the sarcolemma, and that costameres contribute to this pathway, requires confirmation by modern methodologies. It is perhaps significant that the maximum force of contraction is more than half the value estimated for the force of attachment of costameres to the contractile apparatus, suggesting that small changes at costameres might sever those attachments. The roles of many of the proteins at costameres and in the lateral pathway for force transmission are still poorly understood. Nevertheless, one important principle seems apparent (Fig. 4): there is redundancy at nearly every level of the pathway, in the kinds of filaments linking the contractile apparatus to the costameres, in the nature of those linkages, in the structures they form at the membrane, and in the interactions of the membrane with the extracellular matrix. These are all likely to be necessary for the lateral transmission of force to be efficient, without compromising the strength or stability of the muscle fiber. It is therefore not surprising that the absence of key elements of this pathway would lead to muscle weakness or damage associated with muscular dystrophy. Understanding how, and how much, these proteins contribute to lateral force transmission will be important in understanding not only the function of healthy skeletal muscle, but also the molecular basis of many muscular dystrophies and myopathies.

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Acknowledgments

We thank A. O’Neill for preparing the figures, Dr. P. De Deyne and Mr. P. Reed for their useful comments, and our many colleagues whose research on costameres we have summarized here; we regret that space limitations prevented us from citing much of their work. Our research has been supported by grants from the FSH Society, the Muscular Dystrophy Association, and the National Institutes of Health (R21 NS43976 and RO1 NS17282).

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Keywords:

sarcolemma; contraction; dystrophin; cytoskeleton; membrane skeleton; extracellular matrix

©2003 The American College of Sports Medicine