Download Cytoskeletal Disruption After Eccentric Contraction

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
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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
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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-
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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-
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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.
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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
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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
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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
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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
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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
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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
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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.
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