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CLINICAL ORTHOPAEDICS AND RELATED RESEARCH Number 403S, pp. S90–S99 © 2002 Lippincott Williams & Wilkins, Inc. Cytoskeletal Disruption After Eccentric Contraction-Induced Muscle Injury Richard L. Lieber, PhD*; Sameer Shah, PhD*; and Jan Fridén, MD, PhD** Skeletal muscle cytoskeletal proteins are receiving more attention recently based on their importance in maintaining muscle integrity, their role in transmitting force throughout the cell, and their involvement in muscle diseases. In this report, the authors focus on the intermediate filament system of skeletal muscle composed of the protein desmin. Desmin is shown to transmit force from myofibrillar force generators to the muscle surface and to the muscle-tendon junction. This protein is lost rapidly during highintensity exercise using a rabbit model. Mice were genetically engineered that lack the desmin gene and these muscles were shown to generate lower stress but actually to experience less injury during intense exercise. Finally, direct imaging of muscle cells with fluorescently labeled cytoskeletal proteins shows that lack of the desmin protein results in tremendous disorganization of the myofibrillar lattice which may help to explain desmin myopathies. List of Abbreviations Used mRNA messenger ribonucleic acid Athletic and fitness programs are on the rise in the United States. It is estimated that more than 60 million Americans are involved in organized sports and that more than 1⁄2 of American adults exercise regularly.25 This emphasis on fitness has led to a dramatic increase in the number and types of activity-related injuries. In fact, more than 20 million people per year sustain some type of injury severe enough to limit activity in the workplace. Athletic activity in the form of rehabilitation also is increasing the ability of the elderly population to expedite recovery after injury or hospitalization. However, a rational scientific basis for most prescribed strengthening exercises is lacking. This is in large part attributable to the lack of understanding of the basic mechanisms that cause muscle injury and the process of muscular adaptation after injury. The current authors summarize one aspect of a muscle’s response to eccentric exercise (defined as lengthening of an activated muscle). This type of exercise has been shown to have beneficial and deleterious effects on muscle. On the positive side, eccentric exercises provide a potent muscle strengthening stimulus; on the negative side, eccentric exercises specifically From the *Departments of Orthopaedics and Bioengineering, Veterans Affairs Medical Center and University of California, San Diego, CA; and the **Department of Hand Surgery, Göteborg University, Göteborg, Sweden. This work was supported by National Institutes of Health grant AR40050, the Swedish Medical Research Council, and the National Science Foundation. Reprint requests to Richard L. Lieber, PhD, Department of Orthopaedics, U.C. San Diego School of Medicine and V.A. Medical Center, 3350 La Jolla Village Drive, La Jolla, CA 92093–9151. DOI: 10.1097/01.blo.0000031310.06353.9f S90 Number 403S October, 2002 Cytoskeletal Disruption Attributable to Muscle Injury are associated with muscle injury and soreness.2,5,6 This correlation has led many to state, unequivocally, that muscle injury is required for muscle strengthening. Although this statement has anecdotal support based on correlations between injury and strengthening, a causal relationship has not been established. Many investigators also report that eccentric training provides a protective effect against additional eccentric exercise-induced muscle injury.4,19,29 Unfortunately, basic information is lacking regarding the cellular mechanics of eccentric exercise-induced muscle injury, mechanical force transmission within the cell, and there is no explanation for the dramatic protective effect of eccentric exercise against injury that occurs in subsequent eccentric exercise bouts. Therefore, it is problematic to try to apply eccentric exercises therapeutically in the fields of athletics and rehabilitation. Several studies have begun to establish the biologic and mechanical events associated with eccentric contraction-induced injury of skeletal muscle and several reviews are available on this topic.1,5,18,21 The current authors focus on the eccentric exercise effect that relate specifically to the muscle’s cytoskeleton, that is, the structures within the cell not responsible for force generation but rather force transmission. An increased understanding of these structures ultimately may lead to the development of more rational strengthening methods and improving recovery from disuse. Although most micrographs of skeletal muscle show the dominant striation pattern that results primarily from actin and myosin, numerous other proteins also are present within the cell. Specifically, cytoskeletal proteins maintain the structural integrity of the myofibrillar lattice, transmit force generated by actomyosin interaction, and are ubiquitous throughout the muscle cell.20 One study showed that the intracellular cytoskeleton is an important cellular component in muscle cells as already has been shown in many other cell types.22 The most widely studied of the cytoskeletal proteins are those found in the intrasarcomeric region of the muscle. Studies of these two proteins, titin (also S91 called connectin) and nebulin have revolutionized the understanding of muscle passive tension and sarcomere filament assembly (Fig 1). The titin protein is a giant protein (approximately 3 MDa) that extends as one molecule from the M line to the Z disk of the sarcomere. It is modular in nature and therefore very difficult to clone and sequence.9 However, based on synthesis of interdisciplinary studies, it is clear that titin is responsible for much, if not all, of the passive load-bearing properties of muscle and even may account for passive stiffness differences among different muscles. Nebulin also is a very large protein that seems to act as sort of a ruler to regulate thin filament length.10,26 Nebulin is not required for normal muscle contraction because cardiac muscle, which has no nebulin, is able to contract for the lifetime of the animal. In contrast to titin and nebulin which reside within the sarcomere, the intermediate filament network is located outside the sarcomere, surrounding the myofibrils (Fig 1). A most convincing demonstration of this network was presented by Lazarides,11 who prepared sheets of Z disk material from skeletal muscles and showed selective peripheral localization of a protein linking adjacent Z disks. He named this protein desmin (Greek, desmos link) and hypothesized that the adjacent Z disk connections provided mechanical stabilization to the muscle fiber. The three-dimensional nature of this intermediate filament system also was clarified in extraction studies done by Wang and RamirezMitchell27 in which intermediate filament lattice was shown to contain not only struts between adjacent Z disks, but a Z disk structure involving a doublet of rings of intermediate filaments (probably what was seen by Lazarides in his studies of desmin) and as longitudinal arrangements of intermediate filaments around the outside of the sarcomere. These transverse and longitudinal filaments serve to link adjacent myofibrils mechanically and to provide mechanical continuity outside the sarcomere along the myofibrillar length. Although the precise function of this intermediate filament system is not known, it is suggested that it is in- S92 Lieber et al Clinical Orthopaedics and Related Research Fig 1. A diagrammatic representation of the muscle fiber cytoskeletal lattice is shown. Most of these relationships are hypothesized based on in vitro binding studies between proteins, deduced amino acid sequences from cDNA clones, and immunolabeling studies showing colocalization. The variety of connections between structures provide a myriad of possible routes of force transmission and force transduction. volved in the developmental process because it is one of the first muscle-specific proteins to be expressed during development3,8 and because expression of antisense desmin mRNA can affect the development of myoblasts in culture.12,28 Interestingly, transgenic animals completely lacking the desmin gene are viable and fertile but the heavily used muscles of these mice show abnormalities reminiscent of diseased muscles such as loss of myofibrillar regularity, abnormal placement of nuclei, and abnormal appearance of mitochondria.13,17 Intermediate Filament Changes After Eccentric Contraction Desmin Loss is Rapid and Functionally Significant Approximately 10 years ago, during the authors’ initial studies of the acute effects of ec- centric contraction on muscle, it was calculated that the truly unique mechanical events associated with eccentric contraction only occurred during the first 5 to 7 minutes of the eccentric exercise bout.15 After the first few minutes, eccentric contractions simply represented the phenomenon of an isometric contraction and a passive lengthening occurring in parallel. It only was during the first few minutes that eccentric exercise created significant additional force as had been established by muscle physiologists decades earlier.7 The study direction changed from one that previously had focused primarily on fiber type as a critical factor in muscle injury and, instead, focused on other structural proteins within the sarcomere as playing an important role during eccentric contraction. The main focus became the intermediate filament protein, desmin, described previously.14,15 The tremen- Number 403S October, 2002 Cytoskeletal Disruption Attributable to Muscle Injury dous capacity for muscle cells to alter their desmin immunostaining pattern in response to eccentric exercise was shown. These alterations were very rapid (on the order of minutes, Fig 2) and were functionally significant, correlating with the muscle’s isometric tension producing ability (Fig 3). As these studies were done and the role of desmin and other structural proteins in muscle injury was interpreted, it was realized there existed a tremendous lack of information regarding force transmission within and between muscle fibers. This led Patel and Lieber20 to focus on muscle cytoskeletal proteins as central to the normal function of skeletal muscle. Unfortunately, there are few biologic or mechanical data available regarding the role of desmin in normal muscle, which permits the development of testable mechanistic hypotheses. Desmin Null Muscles Generate Less Isometric Stress and Show Less Injury Than Wild Type Muscles In light of the demonstration of the rapid loss in desmin immunostaining during eccentric con- Fig 2. The percentage of muscle fibers within a section devoid of desmin is shown. Presence or absence of desmin was inferred by labeling with an antidesmin primary antibody. Loss of desmin was greater in the extensor digitorum longus, which experienced approximately 25% fiber strain during eccentric treatment compared with the tibialis anterior, which experienced only approximately 10% fiber strain. Loss of desmin was highly functionally significant in both muscles as indicated by measures of maximum tetanic tension. The data are from Reference 16. S93 Fig 3. The relationship between maximum tetanic tension and percentage of desmin negative fibers for the tibialis anterior (open circles) and extensor digitorum longus (filled circles) is shown. In both cases, there was a significant correlation between the two variables suggesting that desmin loss is associated with decreased muscle function. This strong correlation is observed when the injury level is relatively severe as in this model. The data are from Reference 15. traction, analogous studies of eccentric contractions were done on a homozygous desmin null mouse strain.17 This model provided an excellent opportunity to determine whether desmin played a central role during muscle injury and even to understand force transmission in normal muscle.22 Interestingly, desmin knockout muscles generated significantly lower isometric stress (compared with wild type muscles, PreIso in Fig 4). Of course, differences in isometric stress could have been attributable to any of a multitude of factors within the muscle cell other than the specific role played by desmin. However, data were obtained supporting the central role played by desmin in the subsequent experiment, where muscles were subjected to 10 eccentric contractions imposed at 3-minute intervals (Fig 4). After these 10 contractions, b knockout and wild type muscles generated identical levels of stress (approximately 250 kPa) showing less injury to the knockout muscles and providing support for the idea that an eccentrically exercised wild type muscle is mechanically similar to a desmin null muscle.22 One might argue, however, that the decreased injury to the knockout muscles occurred because they were exposed to lower S94 Lieber et al Clinical Orthopaedics and Related Research Fig 4. The time course of isometric stress achieved before, during, and after the eccentric protocol for wild type (open squares) and desmin null (filled circles) muscles is shown. Preeccentric exercise contractions are noted as PreIsol-3, whereas Posteccentric contractions are noted as PostIsol5. Eccentric contractions are noted as EC1–10. The relative decrease in isometric stress of knockout muscles (approximately 9%) is less than the drop in muscles from age-matched animals (approximately 25%). The complete data set was provided previously.22 EC eccentric contraction; PreIso isometric contractions measured before the eccentric contraction protocol; PostIso isometric contractions measured after the eccentric contraction protocol Fig 5. The time course of isometric stress achieved before, during, and after the eccentric protocol is shown. In spite of the fact that the muscles began at the same stress levels, wild type muscles were much more severely injured compared with desmin knockout muscles. In addition to these groups, an old knockout group was tested that showed less injury compared with the old wild type group. The complete data set was provided previously.22 EC eccentric contraction; PreIso isometric contractions measured before the eccentric contraction protocol; PostIso isometric contractions measured after the eccentric contraction protocol stresses during eccentric contraction. To test this idea, the current authors did a similar experiment in which the stress generated by young knockout muscles was matched fortuitously to that generated by older wild type muscles (Fig 5). Stress decreases with age in mice and therefore the older wild type muscles had equal stress as the younger knockout muscles. The results were obvious: older wild type muscles were much more readily injured compared with young knockout muscles despite of the same stresses, fiber length, and mechanical energy absorbed between groups. Therefore, desmin has a significant functional role in normal muscle and in determining muscle’s response to eccentric contraction. The key questions are, of course, how and why? available that can provide mechanistic explanations for our observations. Why should a desmin knockout muscle generate lower stress and experience less injury compared with a wild type muscle? It was hypothesized that the normal interconnections provided by desmin between sarcomeres along and across the muscle fiber permit efficient transfer of mechanical stress from the myofibril to the fiber exterior. In the absence of desmin, the intermyofibrillar connections may be more compliant permitting a greater degree of internal sarcomere shortening and motion, thereby decreasing the efficiency of force transfer across and along the fiber. Similarly, based on the lack of these connections, we supposed that the opposite situation, where strain is transmitted from the fiber exterior to the myofibrils, also could be less efficient. The result would be a decreased sarcomere strain during a fixed degree of lengthening in knockout compared with wild type muscles and therefore, less injury despite the The Myofibrillar Lattice of the Desmin Knockout May Be More Compliant A continuing problem in the interpretation of these experiments is the lack of information Number 403S October, 2002 Cytoskeletal Disruption Attributable to Muscle Injury fact that the energy delivered to the desmin knockout muscle was identical. Preliminary support of these hypotheses has been obtained.22 First, a stereologic method was developed to quantify the longitudinal displacement of Z disks in adjacent myofibrils. Because desmin interconnects these myofibrils, it was hypothesized that a greater displacement would occur in knockout muscles compared with control muscles. In fact, this was the case.22 At resting lengths, displacement of adjacent Z disks was significantly greater in the injured knockout muscles (0.36 0.03 m) compared with wild type muscles (0.22 0.03 m). In a second study, Shah et al23 measured the distance between adjacent Z disks in wild type and knockout muscles that were fixed over a range of sarcomere lengths from approximately 1.8 m to approximately 3.3 m. It was found that, at resting sarcomere length, the stagger between Z disks of adjacent knockout muscles was greater than wild type muscles and that this difference became much more dramatic with increasing sarcomere strain (Fig 6). This large difference was observed even though the wild type muscles were stretched to longer sarcomere lengths (approximately 3.7 m) than the desmin null muscles (approximately 3.3 m). Therefore, if anything, differences between muscle types were underestimated. It was postulated that myofibrils from desmin null muscles are more able to slide relative to one another compared with myofibrils from wild type muscles. This interpretation from electron microscopy has two main limitations. First, inference of a dynamic process such as myofibrillar sliding from static snapshots of myofibrils is a bit weak and, second, no force measurements were available to provide realistic estimates of the actual magnitude of force transmitted in this system. To overcome these limitations, realtime confocal microscopy methods were developed (Fig 7). Desmin Null Fibers Show Greater Sarcomere Disorganization The authors have mechanically tested single fiber segments of wildtype and desmin null S95 Fig 6. Longitudinal displacement of adjacent Z disks stereologically sampled from wild type (circles) and desmin null (triangles) muscles is shown. Displacement was sampled from superficial and deep regions of both muscle types. No difference was observed between regions. The increased displacement of adjacent sarcomeres in the desmin null compared with wild type muscles can be seen. These data were interpreted as indicating the myofibrillar lattice is more compliant in desmin null muscles, although no force was measured directly. The data were published previously.23 fibers. Although wild type fibers have isovolumic behavior, decreasing cross-sectional area with increasing stretch, very little reduction in cross-sectional area with stretch is observed in desmin null fibers, implying that desmin plays a role in constraining the myofibrillar matrix. Qualitative differences in the strain fields also are observed in these fibers in terms of the crispness of the Z disk label and the continuity of the strain field from one edge of the fiber diameter to the other (seen on comparison of Fig 7B with 7D). Desmin Null Fibers Show Lower Eulerian Stress Compared With Wild Type Fibers During Passive Loading Mechanically, as may have been expected from the measured increased myofibrillar sliding (Fig 6), desmin null fibers were more compliant compared with their wild type counterparts. Specifically, at moderate longitudinal (z) strains (z 1.45 or 45% strain), knockout fibers had a slightly lower Eulerian stress compared with wild type fibers (mean standard error of the mean, 18.55 1.78 kPa versus 27.13 3.66 S96 Clinical Orthopaedics and Related Research Lieber et al B A C D Fig 7A–D. Double-labeled confocal images of single muscle fiber segments are shown. (A) Wild type fiber at short sarcomere length is shown. (B) Wild type fiber at long sarcomere length is shown. (C) Desmin null fiber at short sarcomere length is shown. (D) Desmin null fiber at long sarcomere length is shown. Green label represents -actinin, a Z disk protein, whereas red label represents talin, a costameric protein. The desmin null fiber has greater variability in Z disk spacing and does not decrease diameter to as great a degree with passive stretch. All micrographs are at the same magnification, 533. approximately kPa), whereas at higher strains (z 1.75, 75% strain), there was a twofold difference in stress (22.71 4.87 kPa versus 46.98 3.51 kPa). These stresses are reported as Eulerian rather than the traditional Langrangian stresses because the real-time change in fiber cross-sectional area must be taken into account to make proper stress calculations. The fact that desmin null fibers were not isovolumic with stretch was shown by calculating radial strain (r) compared with longitudinal strain. When wild type fibers were stretched, fiber diameter decreased precipitously (at z 1.45, radial strain, r 0.76 0.02 and at z 1.75, r 0.65 0.05). In contrast, desmin null fibers respond to increasing loads in a dramatically different manner, with only a slight decrease in diameter with increasing strain (at z 1.45, r 0.87 0.02 and at z 1.75, r 0.86 0.02). Fourier Analysis of Confocal Images Quantifies Cytoskeletal Periodicity and Regularity To characterize the strain fields and quantify periodicity and relative displacements of the Z disk protein -actinin and the costameric protein talin, a fast fourier transform algorithm was developed to provide a numeric assessment of the fiber. Specifically, confocal images were analyzed using MATLAB (version 6.1, MathWorks, Inc, Indianapolis, IN) as described in detail elsewhere.24 First, the coordinate plane of image was rotated such that the longitudinal axis of the fiber was parallel to the horizontal axis of the imaging coordinate system. Next, the region of analysis was cropped from the original image and dimensioned in pixels. Fast fourier transforms of the intensity profile along each pixel row were calculated, and periodicity details for each row were extracted from the array of power frequency spectra, where frequency is represented in units of inverse sarcomere length or 1/mm. Representative three-dimensional plots of power spectra along the width of the fiber segment are shown in Figure 8. Peak frequencies were inverted to yield spatial periodicity, or sarcomere length. Homogeneity of periodicity then could be assessed by calculating the standard deviation of the periodicity along the Number 403S October, 2002 Cytoskeletal Disruption Attributable to Muscle Injury S97 of the number of elements contributing to that peak. An ancillary goal would be to formulate an explicit development of this methodologic approach to other periodic biologic structures. Preliminary interpretation of the fourier analysis is that the Z disk and costamere periodicities are identical, even at high degrees of stretch (Table 1) and that the coefficient of variation in sarcomere lengths from desmin null fibers is several times that of the wild type. Currently, the coefficient of variation to indicate relative variability is being used. Coefficient of variation (defined as the sample standard deviation divided by the sample mean) is a good indicator in that it is statistically unbiased (does not tend away from the mean with change in sample size) and is understood easily by most scientists. Of course, statistical analysis on the coefficient of variation values must first be arcsin transformed because raw percentages are not distributed normally. These results suggest greater sarcomere length dispersion at long sarcomere lengths in desmin-null fibers. However, with this method, stress can be correlated directly with these spatial heterogeneity measures because the measures can be done on unfixed tissues. Fig 8A–B. Two-dimensional representation of the fast fourier transform of -actinin periodicity across the width of one fiber stretched to sarcomere length approximately 4.5 m. (A) wild type fiber and (B) desmin null muscle fiber are shown. The desmin null fiber shows peaks of lower height and also are more variable in position. width of the fiber. In addition to periodicity, the magnitudes of the power peak height and width were used as a measure of the distinctness of staining, and the relative position of Z disks with respect to each other. If Z disks were confined to the vicinity of adjacent Z disks, the staining pattern would display sharper and more continuous bands of fluorescence across the fiber width, as seen in the wild type fiber (Fig 7). This is because in the frequency domain, the peak intensity is proportional to the square Future Directions and Clinical Significance Based on the popularity of eccentric contractionbiased protocols in exercise and rehabilitation, the ultimate clinical significance of the current work lies in understanding the mechanism of muscle injury and strengthening. There are numerous issues that remain in this field TABLE 1. Sarcomere Length (-actinin) Periodicity of Confocal Images Genotype Wild type Desmin null Slack Fibers Stretched Fibers 3.08 0.049 m (CV, 1.59%) 2.90 0.196 m (CV, 6.76%) 4.37 0.082 m (CV, 1.88%) 4.32 0.219 m (CV, 5.07%) CV coefficient of variation S98 Lieber et al that include but are not limited to questions such as: Is muscle injury actually required for muscle strengthening or is it simply associated with strengthening?; What is the mechanistic connection between muscle tissue stress and upregulation of genes responsible for strengthening?; What is the correlation between mechanical stress, strain and injury or strengthening?; What are the specific cellular components that sense muscle mechanical state?; What are the signaling pathways involved in mechanical transduction and, ultimately, gene upregulation?; and What are the structural adaptations that occur in muscle that protect it from additional injury? The exciting possibility still exists that one of the links in the signal transduction and/or muscle strengthening pathways could be the intermediate filament lattice or perhaps the giant intrasarcomeric protein, titin. Such intriguing possibilities only can be tested once the normal structure and function of these networks is defined. It is incumbent on basic scientists and orthopaedic surgeons who treat muscle injury to maintain a strong network of communication to solve these problems that cost tremendous amounts of money for the American public each year in terms of work days lost, decreased performance, and direct medical costs. References 1. Armstrong RB: Mechanism of exercise-induced delayed onset muscular soreness: A brief review. Med Sci Sports Exerc 16:529–538, 1984. 2. Armstrong RB, Ogilvie RW, Schwane JA: Eccentric exercise-induced injury to rate skeletal muscle. J Appl Physiol 54:80–93, 1983. 3. 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