Evidence for increased myofibrillar mobility in desmin-null mouse skeletal muscle
1 Departments of Orthopaedics and Bioengineering, Veterans Affairs Medical Center and University of California at San Diego, San Diego, CA 92093, USA,
2 Institute of Biomedical Engineering, National Cheng Kung University, Tainan, Taiwan,
3 Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA and
4 Department of Hand Surgery, Sahlgrenska University Hospital, Göteborg, Sweden
*Author for correspondence (e-mail: rlieber{at}ucsd.edu)
Accepted 22 November 2001
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Summary |
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Key words: passive strain, electron microscopy, intermediate filament, force transmission, muscle, mouse.
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Introduction |
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In spite of plentiful information regarding the localization of desmin filaments throughout the muscle cell, there is scant evidence for the mechanical function of the intermediate filament system in skeletal muscle. In ghost muscle fibers, in which the myofibrillar apparatus was chemically extracted, Wang and Ramirez-Mitchell (1983) measured significant mechanical load-bearing by intermediate filaments only at very long sarcomere lengths, over 5 µm. This result suggested that the muscle intermediate filaments may not bear significant loads at physiological sarcomere lengths, which rarely exceed 3.5 µm (Burkholder and Lieber, 2001
). However, indirect evidence suggests that desmin does play an important functional role in normal muscle. Muscles from desmin-null (des /) mice created via homologous recombination (Li et al., 1996
; Milner et al., 1996
) generate a lower isometric stress (Sam et al., 2000
) and exhibit decreased strength (Li et al., 1997
) than muscles from wild-type (+/+) mice. However, paradoxically, in spite of the weakness of these muscles, the desmin-null muscles appear to be protected from the mechanical injury that occurs after a bout of eccentric contractions, even when corrected for the lower stresses generated by the muscles from knockout animals (Sam et al., 2000
). It is difficult to explain such observations on the basis of the immunolocalization and ultrastructural data currently available. Thus, the purpose of the present study was to quantify the longitudinal mobility of myofibrils during muscle extension to elucidate further the functional roles of intermediate filaments in skeletal muscle.
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Materials and methods |
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All procedures were performed in accordance with the NIH Guide for the Use and Care of Laboratory Animals and were approved by the University of California and Department of Veterans Affairs Committees on the Use of Animal Subjects in Research. Each mouse was anesthetized with a cocktail of 10 mg kg1 ketamine, 5 mg kg1 rompum and 1 mg kg1 acepromazine delivered by intraperitoneal injection. The mouse was then killed by intracardiac injection of concentrated sodium pentobarbital. The hindlimbs were skinned and transected below the hip, leaving the entire knee joint intact, and placed for further dissection (within 15 min of death) into a Ringers solution (adjusted to pH 7.5) composed of 137 mmol l1 NaCl, 5 mmol l1 KCl, 1 mmol l1 NaH2PO4, 2 mmol l1 CaCl2, 1 mmol l1 MgSO4 and 11 mmol l1 glucose containing 10 mg l1 curare. Each muscle was then dissected, passively stretched from approximately 100 to approximately 150 % of slack muscle length to generate a range of sarcomere lengths and tied to a wooden applicator.
Stretched muscles were submerged into phosphate-buffered Karnovskys fixative (6 % buffered glutaraldehyde plus formaldehyde), in which they were allowed to incubate overnight at 4°C. Specimens were washed three times in cold (4°C) sodium cacodylate buffer (0.1 mol l1 adjusted to pH 7) and then further fixed in 2 % osmium tetroxide for 1 h at room temperature (23°C). After three buffer washes of 5 min each, muscles were dehydrated in a graded ethanol series and propylene oxide. Specimens were then cut into approximately equal-sized pieces, embedded in Scipoxy 812 Resin (Energy Beam Sciences, Agawam, MA, USA) and oriented to enable longitudinal sectioning. Micrographs were photographed from longitudinal sections of deep and superficial regions (approximately 150 µm2 per micrograph) randomly distributed through the fiber (five micrographs for each depth, giving 10 micrographs per muscle). The sections were confirmed to be in perfect longitudinal alignment by tracking the myofibrils (maximum diameter approximately 0.7 µm) across the entire micrograph (maximum length approximately 14 µm) without observing myofibrils shifting into and out of the section plane.
The end points of all Z-disks from each micrograph were digitized (Fig. 1), allowing the quantification of the horizontal displacement of adjacent Z-disks (xmyo; 4580 measurements per micrograph) and the calculation of sarcomere length (SL; five measurements per micrograph).
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A 10x10 grid of 15 mm squares was printed on a transparency, and coordinates were numbered on each side. The grid was placed at a fixed point on each micrograph, and the structure at each one of the 100 intersecting test points was categorized as myofibril, mitochondria, sarcoplasmic reticulum (SR) or other, where other represented extramyofibrillar structures that could not definitively be categorized as either mitochondria or SR. At test points lying on borders between structures, the component visible in the upper left corner of the intersection was credited to the point in question. Finally, micrographs were resorted into their original groups upon completion of point counting, and the test point fraction of each structure was tabulated, corresponding to the volume density, or volume fraction, of each structure (Weibel, 1980).
An unpaired t-test was used to determine whether horizontal Z-disk displacement and/or sarcomere length were significantly different in deep and superficial regions of muscle fibers, and linear regression was used to measure the association between xmyo and SL in response to passive stretch. The slopes for data from muscles of wild-type versus knockout animals were compared by analysis of covariance (ANCOVA). Differences in misalignment at short (SL<2.20 µm) and long (SL>2.90 µm) sarcomere lengths, corresponding to strains resulting in insignificant and significant passive loads, respectively, were compared by one-way analysis of variance (ANOVA) (Statview 5.0, Abacus Concepts, Inc., Berkeley, CA, USA). The range of sarcomere lengths analyzed also corresponds to the shortest and longest operating sarcomere lengths for the mouse EDL (James et al., 1995
). Values obtained from point counting were compared using a two-way ANCOVA, with genotype (wild-type versus desmin-null) and depth (superficial versus deep) as grouping factors and sarcomere length as the covariate.
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Results |
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No significant differences in myofibrillar, mitochondrial or SR content between muscles from wild-type or desmin-null animals were observed (P>0.6), nor were differences seen as a function of depth (P>0.6) (Table 1). Sarcomere length was noted to be a significant covariate (P<0.01) from ANCOVA, indicating that these values were sensitive to the fixation length.
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Discussion |
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The maximum tethering radius (a term coined to describe the maximum longitudinal extension of the desmin network surrounding a Z-disk) is estimated to be approximately 0.32 µm from the Z-disk displacement in muscles from wild-type animals at high passive strain. This value remained relatively constant as a function of sarcomere length. In contrast, in knockout animals, the value of xmyo increased as a function of sarcomere length and, thus, an upper value for tethering radius cannot be readily inferred (Fig. 2). The existence of a limit on the extension of desmin in wild-type animals suggests a mechanism for the recruitment of desmin into a network of force transmission, whether as a longitudinal load bearer or as a component in a radial force-transmission system. Specifically, desmin could act as a highly compliant or unloaded spring at low extensions, but at increasing extension would exhibit the properties of an extremely stiff spring or, quite possibly, a rigid linkage. Details of this relationship could be elucidated by characterizing the mechanical properties of the desmin filaments themselves. In addition, such a network of filamentous connections, which integrates the entire cytoskeleton, could play an important role in strain-mediated signal transduction, such as that suggested by a study of endothelial cells (Maniotis et al., 1997
). Analogous studies could be envisioned in muscle, in which force- and length-transducing mechanisms could be carefully studied in wild-type or desmin-null mice.
The absence of a tethering radius in muscles from knockout animals argues against other intermediate filaments providing a substitute function for absent desmin. These proteins include those associated with the M-line, such as skelemin (Price, 1987; Price and Gomer, 1993
). These filaments either have a fracture stress less than the passive stress imposed at high strains, and are therefore damaged during increased extension, or are extremely compliant at high strains, and therefore play a minimal role in the structural organization of myofibrils. Finally, from a design standpoint, the absence of a limit to
xmyo in desmin-null muscles provides further evidence against any upregulation of functionally analogous intermediate filament proteins in the knockout system that would mimic desmin function, such as paranemin, synemin or plectin (Carlsson et al., 2000
), since, if these proteins were dramatically upregulated, one might expect less mobility in the myofibrils of muscles from desmin-null animals.
Differences in Z-disk displacement between muscles from wild-type and knockout animals may explain the observation of the lower isometric stress generated by desmin-null muscles as well as their reduced susceptibility to injury that we recently reported (Sam et al., 2000). However, Z-disk displacement was measured in the previous study only from specimens at slack length. Thus, it was not clear whether the differences were permanent, fixed differences between muscles or whether they suggested underlying differences in interconnections between adjacent myofibrils. The present data support the idea of differences in interconnections between muscles of different genotypes. On the basis of the increased mobility observed in desmin-null muscles, sliding of adjacent myofibrils could result in inefficient force transmission due to energy dissipation (explaining the lower isometric stress). Using similar logic, mechanically uncoupling myofibrils during mechanical loading of a muscle fiber could serve a protective role (explaining the decreased muscle injury). It is conceivable that damaged myofibrils without a surrounding desmin intermediate filament lattice (Lieber et al., 1996
) would have an opportunity to repair themselves prior to re-integrating themselves into the load-bearing network. During this myofibrillar repair, muscle function would not be completely compromised since other sarcomeres could transmit force normally. It does not appear that the increase in myofibrillar mobility or decrease in force-generating ability (Sam et al., 2000
) in muscles from desmin-knockout animals is due to inherent differences in myofibrillar content (Table 1). Note, however, that it is possible that stereology of transverse muscle sections may reveal differences in the density of structures involved in the excitationcontraction coupling process.
Finally, the observation of increased Z-disk mobility in desmin-knockout mice at SL well below 5.0 µm, i.e. lengths at which Wang and Ramirez-Mitchell (1983) reported significant passive load-bearing ability in ghost fibers, suggests that the role of intermediate filaments should be re-examined in a system free from the confounding geometrical effects of sarcomere protein extraction. In particular, it is possible that the collapse of the intermediate filament system as a result of myofibrillar extraction altered the mechanical boundary conditions of the intermediate filament lattice during testing, thereby significantly affecting the mechanical properties measured.
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Acknowledgments |
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