A Highlights from MBoC Selection: Nebulin binding impedes mutant desmin filament assembly

Desmin intermediate filaments (DIFs) form an intricate meshwork that organizes myofibers within striated muscle cells. The mechanisms that regulate the association of desmin to sarcomeres and their role in desminopathy are incompletely understood. Here we compare the effect nebulin binding has on the assembly kinetics of desmin and three desminopathy-causing mutant desmin variants carrying mutations in the head, rod, or tail domains of desmin (S46F, E245D, and T453I). These mutants were chosen because the mutated residues are located within the nebulin-binding regions of desmin. We discovered that, although nebulin M160–164 bound to both desmin tetrameric complexes and mature filaments, all three mutants exhibited significantly delayed filament assembly kinetics when bound to nebulin. Correspondingly, all three mutants displayed enhanced binding affinities and capacities for nebulin relative to wild-type desmin. Electron micrographs showed that nebulin associates with elongated normal and mutant DIFs assembled in vitro. Moreover, we measured significantly delayed dynamics for the mutant desmin E245D relative to wild-type desmin in fluorescence recovery after photobleaching in live-cell imaging experiments. We propose a mechanism by which mutant desmin slows desmin remodeling in myocytes by retaining nebulin near the Z-discs. On the basis of these data, we suggest that for some filament-forming desmin mutants, the molecular etiology of desminopathy results from subtle deficiencies in their association with nebulin, a major actin-binding filament protein of striated muscle.     

Structure and function of skeletal muscle in zebrafish early larvae

Zebrafish muscles were examined at an early developmental stage (larvae 5-7 d). Using aluminum clips, preparations (approximately 1.5 mm length, 150 microm diameter) were mounted for force registration and small angle x-ray diffraction. Sarcomeres were oriented mainly in parallel with the preparation long axis. Electrical stimulation elicited fast and reproducible single twitch contractions. Length-force relations showed an optimal sarcomere length of 2.15 microm. X-ray diffraction revealed clear equatorial 1.1/1.0 reflections, showing that myofilaments are predominantly arranged along the preparation long axis. In contrast, reflections from older (2 mo) zebrafish showed two main filament orientations each at an approximately 25 degrees angle relative to the preparation long axis. Electrical stimulation of larvae muscles increased the 1.1/1.0 intensity ratio, reflecting mass transfer to thin filaments during contraction. The apparent lattice volume was 3.42 x 10(-3) microm(3), which is smaller than that of mammalian striated muscle and more similar to that of frog muscles. The relation between force and stimulation frequency showed fusion of responses at a comparatively high frequency (approximately 186 Hz), reflecting a fast muscle phenotype. Inhibition of fast myosin with N-benzyl-p-toluene sulphonamide (BTS) showed that the later phase of the tetanus was less affected than the initial peak. This suggests that, although the main contractile phenotype is fast, slow twitch fibers can contribute to sustained contraction. A fatigue stimulation protocol with repeated 220 ms/186 Hz tetani showed that tetanic force decreased to 50% at a train rate of 0.1 s(-1). In conclusion, zebrafish larvae muscles can be examined in vitro using mechanical and x-ray methods. The muscles and myofilaments are mainly orientated in parallel with the larvae long axis and exhibit a significant fast contractile component. Sustained contractions can also involve a small contribution from slower muscle types.     

Micromechanical function of myofibrils isolated from skeletal and cardiac muscles of the zebrafish

The zebrafish is a potentially important and cost-effective model for studies of development, motility, regeneration, and inherited human diseases. The object of our work was to show whether myofibrils isolated from zebrafish striated muscle represent a valid subcellular contractile model. These organelles, which determine contractile function in muscle, were used in a fast kinetic mechanical technique based on an atomic force probe and video microscopy. Mechanical variables measured included rate constants of force development (k(ACT)) after Ca(2+) activation and of force decay (τ(REL)(-1)) during relaxation upon Ca(2+) removal, isometric force at maximal (F(max)) or partial Ca(2+) activations, and force response to an external stretch applied to the relaxed myofibril (F(pass)). Myotomal myofibrils from larvae developed greater active and passive forces, and contracted and relaxed faster than skeletal myofibrils from adult zebrafish, indicating developmental changes in the contractile organelles of the myotomal muscles. Compared with murine cardiac myofibrils, measurements of adult zebrafish ventricular myofibrils show that k(ACT), F(max), Ca(2+) sensitivity of the force, and F(pass) were comparable and τ(REL)(-1) was smaller. These results suggest that cardiac myofibrils from zebrafish, like those from mice, are suitable contractile models to study cardiac function at the sarcomeric level. The results prove the practicability and usefulness of mechanical and kinetic investigations on myofibrils isolated from larval and adult zebrafish muscles. This novel approach for investigating myotomal and myocardial function in zebrafish at the subcellular level, combined with the powerful genetic manipulations that are possible in the zebrafish, will allow the investigation of the functional primary consequences of human disease-related mutations in sarcomeric proteins in the zebrafish model.     

Characterization of a zebrafish (Danio rerio) desmin cDNA: an early molecular marker of myogenesis

Desmin is a muscle-specific protein and a constitutive subunit of the intermediate filaments (IF) in skeletal, cardiac and smooth muscles. It is an early marker of skeletal muscle myogenesis. We have characterized a clone of desmin cDNA from an embryonic zebrafish (Danio rerio) cDNA library. The full-length cDNA comprised 1798 nucleotides, encoding a protein of 473 amino acids. The predicted amino acid sequence of the zebrafish desmin shares a high degree of similarity to other vertebrate desmins, but also contains a sequence at the carboxyl terminal of the tail domain that is unique to the zebrafish. It carries many features which are distinctive of IF subunit proteins. These include the T/SSYRRXF/Y motif in the head domain, and the intermediate filament signature consensus, [I/V]-X-[T/A/C/I]-Y-[R/K/H]-X-[L/M]-L-[D/E], located in the carboxyl terminus of the central helical rod. Unlike other 3' UTR sequences, the 3' UTR of the zebrafish cDNA sequence has two CAYUG elements flanking a single polyadenylation site. The temporal and spatial expression patterns of desmin mRNA during early zebrafish development were studied. The onset of desmin expression occurred at the 1-3 somite stage (11 hpf). It increased throughout somitogenesis, with maximum expression at the Prim-6 stage (25 hpf), and decreasing expression towards the protruding-mouth stage (72 hpf). Desmin mRNA was initially localised exclusively to the somites, but was subsequently also detected in other musculature in the developing heart and fins. The onset of expression and the spatial localization of desmin mRNA in the zebrafish coincides with that reported for MyoD and myogenin.     

Desmin Is Essential for the Tensile Strength and Integrity of Myofibrils but Not for Myogenic Commitment, Differentiation, and Fusion of Skeletal Muscle

A null mutation was introduced into the mouse desmin gene by homologous recombination. The desmin knockout mice (Des −/−) develop normally and are fertile. However, defects were observed after birth in skeletal, smooth, and cardiac muscles (Li, Z., E. Colucci-Guyon, M. Pincon-Raymond, M. Mericskay, S. Pournin, D. Paulin, and C. Babinet. 1996. Dev. Biol. 175:362–366; Milner, D.J., G. Weitzer, D. Tran, A. Bradley, and Y. Capetanaki. 1996. J. Cell Biol. 134:1255– 1270). In the present study we have carried out a detailed analysis of somitogenesis, muscle formation, maturation, degeneration, and regeneration in Des −/− mice. Our results demonstrate that all early stages of muscle differentiation and cell fusion occur normally. However, after birth, modifications were observed essentially in weight-bearing muscles such as the soleus or continually used muscles such as the diaphragm and the heart. In the absence of desmin, mice were weaker and fatigued more easily. The lack of desmin renders these fibers more susceptible to damage during contraction. We observed a process of degeneration of myofibers, accompanied by macrophage infiltration, and followed by a process of regeneration. These cycles of degeneration and regeneration resulted in a relative increase in slow myosin heavy chain (MHC) and decrease in fast MHC. Interestingly, this second wave of myofibrillogenesis during regeneration was often aberrant and showed signs of disorganization. Subsarcolemmal accumulation of mitochondria were also observed in these muscles. The lack of desmin was not compensated by an upregulation of vimentin in these mice either during development or regeneration. Absence of desmin filaments within the sarcomere does not interfere with primary muscle formation or regeneration. However, myofibrillogenesis in regenerating fibers is often abortive, indicating that desmin may be implicated in this repair process. The results presented here show that desmin is essential to maintain the structural integrity of highly solicited skeletal muscle.     

Two Desmin Gene Mutations Associated with Myofibrillar Myopathies in Polish Families

Desmin is a muscle-specific intermediate filament protein which forms a network connecting the sarcomere, T tubules, sarcolemma, nuclear membrane, mitochondria and other organelles. Mutations in the gene coding for desmin (DES) cause skeletal myopathies often combined with cardiomyopathy, or isolated cardiomyopathies. The molecular pathomechanisms of the disease remain ambiguous. Here, we describe and comprehensively characterize two DES mutations found in Polish patients with a clinical diagnosis of desminopathy. The study group comprised 16 individuals representing three families. Two mutations were identified: a novel missense mutation (Q348P) and a small deletion of nine nucleotides (A357_E359del), previously described by us in the Polish population. A common ancestry of all the families bearing the A357_E359del mutation was confirmed. Both mutations were predicted to be pathogenic using a bioinformatics approach, including molecular dynamics simulations which helped to rationalize abnormal behavior at molecular level. To test the impact of the mutations on DES expression and the intracellular distribution of desmin muscle biopsies were investigated. Elevated desmin levels as well as its atypical localization in muscle fibers were observed. Additional staining for M-cadherin, α-actinin, and myosin heavy chains confirmed severe disruption of myofibrill organization. The abnormalities were more prominent in the Q348P muscle, where both small atrophic fibers as well large fibers with centrally localized nuclei were observed. We propose that the mutations affect desmin structure and cause its aberrant folding and subsequent aggregation, triggering disruption of myofibrils organization.     

Impact of disease mutations on the desmin filament assembly process

It has been documented that mutations in the human desmin gene lead to a severe type of myofibrillar myopathy, termed more specifically desminopathy, which affects cardiac and skeletal as well as smooth muscle. We showed recently that 14 recombinant versions of these disease-causing desmin variants, all involving single amino acid substitutions in the alpha-helical rod domain, interfere with in vitro filament formation at distinct stages of the assembly process. We now provide mechanistic details of how these mutations affect the filament assembly process by employing analytical ultracentrifugation, time-lapse electron microscopy of negatively stained and glycerol-sprayed/low-angle rotary metal-shadowed samples, quantitative scanning transmission electron microscopy, and viscometric studies. In particular, the soluble assembly intermediates of two of the mutated proteins exhibit unusually high s-values, compatible with octamers and other higher-order complexes. Moreover, several of the six filament-forming mutant variants deviated considerably from wild-type desmin with respect to their filament diameters and mass-per-length values. In the heteropolymeric situation with wild-type desmin, four of the mutant variants caused a pronounced "hyper-assembly", when assayed by viscometry. This indicates that the various mutations may cause abortion of filament formation by the mutant protein at distinct stages, and that some of them interfere severely with the assembly of wild-type desmin. Taken together, our findings provide novel insights into the basic intermediate filament assembly mechanisms and offer clues as to how amino acid changes within the desmin rod domain may interfere with the normal structural organization of the muscle cytoskeleton, eventually leading to desminopathy.     

Angiotensin 1–7 prevents the excessive force loss resulting from 14- and 28-day denervation in mouse EDL and soleus muscle

Denervation leads to muscle atrophy, which is described as muscle mass and force loss, the latter exceeding expectation from mass loss. The objective of this study was to determine the efficiency of angiotensin (Ang) 1–7 at reducing muscle atrophy in mouse extensor digitorum longus (EDL) and soleus following 14- and 28-d denervation periods. Some denervated mice were treated with Ang 1–7 or diminazene aceturate (DIZE), an ACE2 activator, to increase Ang 1–7 levels. Ang 1–7/DIZE treatment had little effect on muscle mass loss and fiber cross-sectional area reduction. Ang 1–7 and DIZE fully prevented the loss of tetanic force normalized to cross-sectional area and accentuated the increase in twitch force in denervated muscle. However, they did not prevent the shift of the force–frequency relationship toward lower stimulation frequencies. The Ang 1–7/DIZE effects on twitch and tetanic force were completely blocked by A779, a MasR antagonist, and were not observed in MasR^−/− muscles. Ang 1–7 reduced the extent of membrane depolarization, fully prevented the loss of membrane excitability, and maintained the action potential overshoot in denervated muscles. Ang 1–7 had no effect on the changes in α-actin, myosin, or MuRF-1, atrogin-1 protein content or the content of total or phosphorylated Akt, S6, and 4EPB. This is the first study that provides evidence that Ang 1–7 maintains normal muscle function in terms of maximum force and membrane excitability during 14- and 28-d periods after denervation.     

Desmin integrates the three-dimensional mechanical properties of muscles

Striated muscle is a linear motor whose properties have been defined in terms of uniaxial structures. The question addressed here is what contribution is made to the properties of this motor by extramyofilament cytoskeletal structures that are not aligned in parallel with the myofilaments. This question arose from observations that transverse loads increase muscle force production in diaphragm but not in the hindlimb muscle, thereby indicating the presence of structures that couple longitudinal and transverse properties of diaphragmatic muscle. Furthermore, we find that the diaphragms of null mutants for the cytoskeletal protein desmin show 1) significant reductions in coupling between the longitudinal and transverse properties, indicating for the first time a role for a specific protein in integrating the three-dimensional mechanical properties of muscle, 2) significant reductions in the stiffness and viscoelasticity of muscle, and 3) significant increases in tetanic force production. Thus desmin serves a complex mechanical function in diaphragm muscle by contributing both to passive stiffness and viscoelasticity and to modulation of active force production in a three-dimensional structural network. Our finding changes the paradigm of force transmission among cells by placing our understanding of the function of the cytoskeleton in the context of the structural and mechanical complexity of muscles.     

Locomotor function shapes the passive mechanical properties and operating lengths of muscle

Locomotor muscles often perform diverse roles, functioning as motors that produce mechanical energy, struts that produce force and brakes that dissipate mechanical energy. In many vertebrate muscles, these functions are not mutually exclusive and a single muscle often performs a range of mechanically diverse tasks. This functional diversity has obscured the relationship between a muscle''s locomotor function and its mechanical properties. I use hopping in toads as a model system for comparing muscles that primarily produce mechanical energy with muscles that primarily dissipate mechanical energy. During hopping, hindlimb muscles undergo active shortening to produce mechanical energy and propel the animal into the air, whereas the forelimb muscles undergo active lengthening to dissipate mechanical energy during landing. Muscles performing distinct mechanical functions operate on different regions of the force–length curve. These findings suggest that a muscle''s operating length may be shaped by potential trade-offs between force production and sarcomere stability. In addition, the passive force–length properties of hindlimb and forelimb muscles vary, suggesting that passive stiffness functions to restrict the muscle''s operating length in vivo. These results inform our understanding of vertebrate muscle variation by providing a clear link between a muscle''s locomotor function and its mechanical properties.     

Desmin knockout muscles generate lower stress and are less vulnerable to injury compared with wild-type muscles

The functional roleof the skeletal muscle intermediate filament system was investigated bymeasuring the magnitude of muscle force loss after cyclic eccentriccontraction (EC) in normal and desmin null mouse extensor digitorumlongus muscles. Isometric stress generated was significantly greater inwild-type (313 ± 8 kPa) compared with knockout muscles (276 ± 13 kPa) before EC (P < 0.05), but 1 h after 10 ECs, both muscle types generated identical levels of stress (~250kPa), suggesting less injury to the knockout. Differences in injurysusceptibility were not explained by the different absolute stresslevels imposed on wild-type versus knockout muscles (determined bytesting older muscles) or by differences in fiber length or mechanicalenergy absorbed. Morphometric analysis of longitudinal electronmicrographs indicated that Z disks from knockout muscles were morestaggered (0.36 ± 0.03 µm) compared with wild-type muscles(0.22 ± 0.03 µm), which may indicate that the knockoutcytoskeleton is more compliant. These data demonstrate that lack of theintermediate filament system decreases isometric stress production andthat the desmin knockout muscle is less vulnerable to mechanical injury.

     

Desmin filaments influence myofilament spacing and lateral compliance of slow skeletal muscle fibers

Intermediate filaments composed of desmin interlink Z-disks and sarcolemma in skeletal muscle. Depletion of desmin results in lower active stress of smooth, cardiac, and skeletal muscles. Structural functions of intermediate filaments in fast (psoas) and slow (soleus) skeletal muscle were examined using x-ray diffraction on permeabilized muscle from desmin-deficient mice (Des-/-) and controls (Des+/+). To examine lateral compliance of sarcomeres and cells, filament distances and fiber width were measured during osmotic compression with dextran. Equatorial spacing (x-ray diffraction) of contractile filaments was wider in soleus Des-/- muscle compared to Des+/+, showing that desmin is important for maintaining lattice structure. Osmotic lattice compression was similar in Des-/- and Des+/+. In width measurements of single fibers and bundles, Des-/- soleus were more compressed by dextran compared to Des+/+, showing that intermediate filaments contribute to whole-cell compliance. For psoas fibers, both filament distance and cell compliance were similar in Des-/- and Des+/+. We conclude that desmin is important for stabilizing sarcomeres and maintaining cell compliance in slow skeletal muscle. Wider filament spacing in Des-/- soleus cannot, however, explain the lower active stress, but might influence resistance to stretch, possibly minimizing stretch-induced cell injury.     

Swim-training changes the spatio-temporal dynamics of skeletogenesis in zebrafish larvae (Danio rerio)

Fish larvae experience many environmental challenges during development such as variation in water velocity, food availability and predation. The rapid development of structures involved in feeding, respiration and swimming increases the chance of survival. It has been hypothesized that mechanical loading induced by muscle forces plays a role in prioritizing the development of these structures. Mechanical loading by muscle forces has been shown to affect larval and embryonic bone development in vertebrates, but these investigations were limited to the appendicular skeleton. To explore the role of mechanical load during chondrogenesis and osteogenesis of the cranial, axial and appendicular skeleton, we subjected zebrafish larvae to swim-training, which increases physical exercise levels and presumably also mechanical loads, from 5 until 14 days post fertilization. Here we show that an increased swimming activity accelerated growth, chondrogenesis and osteogenesis during larval development in zebrafish. Interestingly, swim-training accelerated both perichondral and intramembranous ossification. Furthermore, swim-training prioritized the formation of cartilage and bone structures in the head and tail region as well as the formation of elements in the anal and dorsal fins. This suggests that an increased swimming activity prioritized the development of structures which play an important role in swimming and thereby increasing the chance of survival in an environment where water velocity increases. Our study is the first to show that already during early zebrafish larval development, skeletal tissue in the cranial, axial and appendicular skeleton is competent to respond to swim-training due to increased water velocities. It demonstrates that changes in water flow conditions can result into significant spatio-temporal changes in skeletogenesis.     

Lack of desmin results in abortive muscle regeneration and modifications in synaptic structure.

Desmin, a muscle-specific intermediate filament protein, is expressed in all muscle tissues. Its absence leads to a multisystemic disorder involving cardiac, skeletal, and smooth muscles. In skeletal muscle, structural abnormalities include lack of alignment of myofibrils, Z disk streaming, and focal muscle degeneration. In this study, we have examined the consequences of an absence of desmin on the mechanisms of regeneration and the integrity of the neuromuscular junction. The muscles of desmin knock-out and wild-type mice were made to regenerate by injecting cardiotoxin and were examined 7 to 42 days following the injection. The absence of desmin resulted in a delayed and modified regeneration and an accumulation of adipocytes. This was associated with a persistence of small diameter muscle fibers containing both N-CAM and developmental myosin isoforms. The amount of the slow myosin was increased, whereas there was a decrease in the fast isoform in the regenerated muscles of desmin knock-out mice. Both regeneration and aging led to the appearance of elongated neuromuscular junctions with diffuse acetylcholinesterase staining and a decrease in the overall acetylcholinesterase activity in the muscles of these mice. The neuromuscular junctions were markedly disorganised and in some cases postjunctional folds were absent. We conclude that desmin is essential for terminal muscle regeneration, maturation of muscle fibers, and maintaining the complex folded structure of the postsynaptic apparatus of the neuromuscular junctions.     

The series elastic shock absorber: tendon elasticity modulates energy dissipation by muscle during burst deceleration

During downhill running, manoeuvring, negotiation of obstacles and landings from a jump, mechanical energy is dissipated via active lengthening of limb muscles. Tendon compliance provides a ‘shock-absorber’ mechanism that rapidly absorbs mechanical energy and releases it more slowly as the recoil of the tendon does work to stretch muscle fascicles. By lowering the rate of muscular energy dissipation, tendon compliance likely reduces the risk of muscle injury that can result from rapid and forceful muscle lengthening. Here, we examine how muscle–tendon mechanics are modulated in response to changes in demand for energy dissipation. We measured lateral gastrocnemius (LG) muscle activity, force and fascicle length, as well as leg joint kinematics and ground-reaction force, as turkeys performed drop-landings from three heights (0.5–1.5 m centre-of-mass elevation). Negative work by the LG muscle–tendon unit during landing increased with drop height, mainly owing to greater muscle recruitment and force as drop height increased. Although muscle strain did not increase with landing height, ankle flexion increased owing to increased tendon strain at higher muscle forces. Measurements of the length–tension relationship of the muscle indicated that the muscle reached peak force at shorter and likely safer operating lengths as drop height increased. Our results indicate that tendon compliance is important to the modulation of energy dissipation by active muscle with changes in demand and may provide a mechanism for rapid adjustment of function during deceleration tasks of unpredictable intensity.     

Knockdown of fast skeletal myosin-binding protein C in zebrafish results in a severe skeletal myopathy

Myosin-binding protein C (MyBPC) in the muscle sarcomere interacts with several contractile and structural proteins. Mutations in the cardiac isoform (MyBPC-3) in humans, or animal knockout, are associated with cardiomyopathy. Function of the fast skeletal isoform (MyBPC-2) in living muscles is less understood. This question was addressed using zebrafish models, combining gene expression data with functional analysis of contractility and small-angle x-ray diffraction measurements of filament structure. Fast skeletal MyBPC-2B, the major isoform, was knocked down by >50% using morpholino antisense nucleotides. These morphants exhibited a skeletal myopathy with elevated apoptosis and up-regulation of factors associated with muscle protein degradation. Morphant muscles had shorter sarcomeres with a broader length distribution, shorter actin filaments, and a wider interfilament spacing compared with controls, suggesting that fast skeletal MyBPC has a role in sarcomere assembly. Active force was reduced more than expected from the decrease in muscle size, suggesting that MyBPC-2 is required for optimal force generation at the cross-bridge level. The maximal shortening velocity was significantly increased in the MyBPC-2 morphants, but when related to the sarcomere length, the difference was smaller, reflecting that the decrease in MyBPC-2B content and the resulting myopathy were accompanied by only a minor influence on filament shortening kinetics. In the controls, equatorial patterns from small-angle x-ray scattering revealed that comparatively few cross-bridges are attached (as evaluated by the intensity ratio of the 11 and 10 equatorial reflections) during active contraction. X-ray scattering data from relaxed and contracting morphants were not significantly different from those in controls. However, the increase in the 11:10 intensity ratio in rigor was lower compared with that in controls, possibly reflecting effects of MyBPC on the cross-bridge interactions. In conclusion, lack of MyBPC-2 results in a severe skeletal myopathy with structural changes and muscle weakness.     

Role of vimentin in smooth muscle force development

Vimentin intermediate filaments undergo spatial reorganization in cultured smooth muscle cells in response to contractile activation; however, the role of vimentin in the physiological properties of smooth muscle has not been well elucidated. Tracheal smooth muscle strips were loaded with antisense oligonucleotides (ODNs) against vimentin and then cultured for 2 days to allow for protein degradation. Treatment with vimentin antisense, but not sense, ODNs suppressed vimentin protein expression; neither vimentin antisense nor sense ODNs affected protein levels of desmin and actin. Force development in response to ACh stimulation or KCl depolarization was lower in vimentin-deficient tissues than in vimentin sense ODN- or non-ODN-treated muscle strips. Passive tension was also depressed in vimentin-depleted muscle tissues. Vimentin downregulation did not attenuate increases in myosin light chain (MLC) phosphorylation in response to contractile stimulation or basal MLC phosphorylation. In vimentin sense ODN-treated or non-ODN-treated smooth muscle strips, the desmosomal protein plakoglobin was primarily localized in the cell periphery. The membrane-associated localization of plakoglobin was reduced in vimentin-depleted muscle tissues. These studies suggest that vimentin filaments play an important role in mediating active force development and passive tension, which are not regulated by MLC phosphorylation. Vimentin downregulation impairs the structural organization of desmosomes, which may be associated with the decrease in force development. intermediate filaments; cytoskeleton; contraction; desmin     

Theoretical Predictions of the Effects of Force Transmission by Desmin on Intersarcomere Dynamics

Desmin is an intermediate filament protein in skeletal muscle that forms a meshlike network around Z-disks. A model of a muscle fiber was developed to investigate the mechanical role of desmin. A two-dimensional mesh of viscoelastic sarcomere elements was connected laterally by elastic elements representing desmin. The equations of motion for each sarcomere boundary were evaluated at quasiequilibrium to determine sarcomere stresses and strains. Simulations of passive stretch and fixed-end contractions yielded values for sarcomere misalignment and stress in wild-type and desmin null fibers. Passive sarcomere misalignment increased nonlinearly with fiber strain in both wild-type and desmin null simulations and was significantly larger without desmin. During fixed-end contraction, desmin null simulations also demonstrated greater sarcomere misalignment and reduced stress production compared with wild-type. In simulations with only a fraction of wild-type desmin present, fixed-end stress increased as a function of desmin concentration and this relationship was influenced by the cellular location of the desmin filaments. This model suggests that desmin stabilizes Z-disks and enables greater stress production by providing a mechanical tether between adjacent myofibrils and to the extracellular matrix and that the significance of the tether is a function of its location within the cell.     

Passive skeletal muscle can function as an osmotic engine

Muscles are composite structures. The protein filaments responsible for force production are bundled within fluid-filled cells, and these cells are wrapped in ordered sleeves of fibrous collagen. Recent models suggest that the mechanical interaction between the intracellular fluid and extracellular collagen is essential to force production in passive skeletal muscle, allowing the material stiffness of extracellular collagen to contribute to passive muscle force at physiologically relevant muscle lengths. Such models lead to the prediction, tested here, that expansion of the fluid compartment within muscles should drive forceful muscle shortening, resulting in the production of mechanical work unassociated with contractile activity. We tested this prediction by experimentally increasing the fluid volumes of isolated bullfrog semimembranosus muscles via osmotically hypotonic bathing solutions. Over time, passive muscles bathed in hypotonic solution widened by 16.44 ± 3.66% (mean ± s.d.) as they took on fluid. Concurrently, muscles shortened by 2.13 ± 0.75% along their line of action, displacing a force-regulated servomotor and doing measurable mechanical work. This behaviour contradicts the expectation for an isotropic biological tissue that would lengthen when internally pressurized, suggesting a functional mechanism analogous to that of engineered pneumatic actuators and highlighting the significance of three-dimensional force transmission in skeletal muscle.