Glycosaminoglycans contribute to extracellular matrix fiber recruitment and arterial wall mechanics

Elastic and collagen fibers are well known to be the major load-bearing extracellular matrix (ECM) components of the arterial wall. Studies of the structural components and mechanics of arterial ECM generally focus on elastin and collagen fibers, and glycosaminoglycans (GAGs) are often neglected. Although GAGs represent only a small component of the vessel wall ECM, they are considerably important because of their diverse functionality and their role in pathological processes. The goal of this study was to study the mechanical and structural contributions of GAGs to the arterial wall. Biaxial tensile testing was paired with multiphoton microscopic imaging of elastic and collagen fibers in order to establish the structure–function relationships of porcine thoracic aorta before and after enzymatic GAG removal. Removal of GAGs results in an earlier transition point of the nonlinear stress–strain curves \((p<0.05)\). However, stiffness was not significantly different after GAG removal treatment, indicating earlier but not absolute stiffening. Multiphoton microscopy showed that when GAGs are removed, the adventitial collagen fibers are straighter, and both elastin and collagen fibers are recruited at lower levels of strain, in agreement with the mechanical change. The amount of stress relaxation also decreased in GAG-depleted arteries \((p<0.05)\). These findings suggest that the interaction between GAGs and other ECM constituents plays an important role in the mechanics of the arterial wall, and GAGs should be considered in addition to elastic and collagen fibers when studying arterial function.     

Sarcomere length dispersion in single skeletal muscle fibers and fiber bundles.

Light diffraction patterns produced by single skeletal muscle fibers and small fiber bundles of Rana pipiens semitendinosus have been examined at rest and during tetanic contraction. The muscle diffraction patterns were recorded with a vidicon camera interfaced to a minicomputer. Digitized video output was analyzed on-line to determine mean sarcomere length, line intensity, and the distribution of sarcomere lengths. The occurrence of first-order line intensity and peak amplitude maxima at approximately 3.0 mum is interpreted in terms of simple scattering theory. Measurements made along the length of a singel fiber reveal small variations in calculated mean sarcomere length (SD about 1.2%) and its percent dispersion (2.1% +/- 0.8%). Dispersion in small multifiber preparations increases approximately linearly with fiber number (about 0.2% per fiber) to a maximum of 8-10% in large bundles. Dispersion measurements based upon diffraction line analysis are comparable to SDs calculated from length distribution histograms obtained by light micrography of the fiber. First-order line intensity decreases by about 40% during tetanus; larger multifibered bundles exhibit substantial increases in sarcomere dispersion during contraction, but single fibers show no appreciable dispersion change. These results suggest the occurrence of asynchronous static or dynamic axial disordering of thick filaments, with a persistence in long range order of sarcomere spacing during contraction in single fibers.     

Zebrafish periostin is required for the adhesion of muscle fiber bundles to the myoseptum and for the differentiation of muscle fibers

The myoseptum of fishes, composed of dense collagen, is a connective tissue layer that forms in the embryo, dividing somites from the trunk, and its structure and function are similar to those of the mammalian tendon. Both the myoseptum and tendon serve as the transmitter of muscular contractility to bones and adjoining muscles, and their structure is indispensable for movement of vertebrate animals. We cloned the zebrafish periostin gene and examined its expression and function in the myoseptum. The expression in embryos started in the rostral part of each segmented somite in the early segmentation stage; and consequently, metameric stripes were observed. At the end of segmentation, the expression region shifted to the transverse myoseptum and the myotome-epidermis boundary, and each myotome was surrounded by periostin. Using a polyclonal antibody, we found that the periostin protein was localized to the transverse myoseptum. Consistently, periostin morpholino antisense oligonucleotide led to defects in myoseptum formation, a delay in the differentiation of myofibers, and disorder of connection between myofibrils and myoseptum. We demonstrated here that periostin is the first molecule involved in myoseptum formation and propose that periostin secretion on the surface of the myoseptum is required for the adhesion of muscle fiber bundles to the myoseptum and the differentiation of muscle fibers.     

From single muscle fiber to whole muscle mechanics: a finite element model of a muscle bundle with fast and slow fibers

Muscles exhibit highly complex, multi-scale architecture with thousands of muscle fibers, each with different properties, interacting with each other and surrounding connective structures. Consequently, the results of single-fiber experiments are scarcely linked to the macroscopic or whole muscle behavior. This is especially true for human muscles where it would be important to understand of how skeletal muscles disorders affect patients’ life. In this work, we developed a mathematical model to study how fast and slow muscle fibers, well characterized in single-fiber experiments, work and generate together force and displacement in muscle bundles. We characterized the parameters of a Hill-type model, using experimental data on fast and slow single human muscle fibers, and comparing experimental data with numerical simulations obtained from finite element (FE) models of single fibers. Then, we developed a FE model of a bundle of 19 fibers, based on an immunohistochemically stained cross section of human diaphragm and including the corresponding properties of each slow or fast fiber. Simulations of isotonic contractions of the bundle model allowed the generation of its apparent force–velocity relationship. Although close to the average of the force–velocity curves of fast and slow fibers, the bundle curve deviates substantially toward the fast fibers at low loads. We believe that the present model and the characterization of the force–velocity curve of a fiber bundle represents the starting point to link the single-fiber properties to those of whole muscle with FE application in phenomenological models of human muscles.     

Maturational changes in extracellular matrix and lung tissue mechanics.

The viscoelastic properties of the pulmonary parenchyma change rapidly postparturition. We compared changes in mechanical properties with changes in tissue composition of rat lung parenchymal strips in three groups of Sprague-Dawley rats: baby (B; 10-14 days), young (Y; approximately 3 wk), and adult (A; approximately 8 wk). Strips were suspended in an organ bath, and resistance (R), elastance (E), and hysteresivity (eta) were calculated during sinusoidal oscillations before and after the addition of acetylcholine (ACh) (10(-3) M). Strips were then fixed in formalin, and sections were stained with hematoxylin and eosin, Verhoff's elastic stain, or Van Gieson's picric acid-fuchsin stain for collagen. The volume proportion of collagen (%Col), the length density of elastic fibers (L(V)/Pr(alv)), and the arithmetic mean thickness of alveolar septae (T(a)) were calculated by morphometry. Tissue was also stained for alpha-smooth muscle actin (ASMA), and the volume proportion of ASMA (%ASMA) was calculated. Hyaluronic acid (HA) was quantitated by radioimmunoassay in separate strips. R and E in B strips were significantly higher, whereas eta was significantly smaller than in Y or A strips. Changes in these parameters with ACh were greater in B strips. T(a), %ASMA, and HA were greatest in B strips, whereas %Col and L(V)/Pr(alv) were least. There were significant positive correlations between R and E vs. T(a) and between percent change in R and eta post-ACh vs. T(a) and vs. %ASMA, and significant negative correlations between R and E vs. %Col and vs. L(V)/Pr(alv) and percent increase in all three mechanical parameters post-ACh vs. %Col. These data suggest that the relatively high stiffness, R, and contractile responsiveness of parenchymal tissues observed in newborns are not directly attributable to the amount of collagen and elastic fibers in the tissue, but rather they are related to the thickened alveolar wall and the relatively greater percent of contractile cells.     

Lateral mechanics of muscle fibers and its role in signaling

The minireview considers some aspects of the lateral mechanics of muscle fibers, such as the available data on transverse stiffness of intact muscle cells, skinned fibers, and isolated myofibrils under various conditions and at different differentiation stages; the mechanisms whereby extrasarcomeric cytoskeletal proteins are involved in forming the structural basis of transverse stiffness; and their possible signaling role.     

Viscoelastic retraction of single living stress fibers and its impact on cell shape, cytoskeletal organization, and extracellular matrix mechanics

Cells change their form and function by assembling actin stress fibers at their base and exerting traction forces on their extracellular matrix (ECM) adhesions. Individual stress fibers are thought to be actively tensed by the action of actomyosin motors and to function as elastic cables that structurally reinforce the basal portion of the cytoskeleton; however, these principles have not been directly tested in living cells, and their significance for overall cell shape control is poorly understood. Here we combine a laser nanoscissor, traction force microscopy, and fluorescence photobleaching methods to confirm that stress fibers in living cells behave as viscoelastic cables that are tensed through the action of actomyosin motors, to quantify their retraction kinetics in situ, and to explore their contribution to overall mechanical stability of the cell and interconnected ECM. These studies reveal that viscoelastic recoil of individual stress fibers after laser severing is partially slowed by inhibition of Rho-associated kinase and virtually abolished by direct inhibition of myosin light chain kinase. Importantly, cells cultured on stiff ECM substrates can tolerate disruption of multiple stress fibers with negligible overall change in cell shape, whereas disruption of a single stress fiber in cells anchored to compliant ECM substrates compromises the entire cellular force balance, induces cytoskeletal rearrangements, and produces ECM retraction many microns away from the site of incision; this results in large-scale changes of cell shape (> 5% elongation). In addition to revealing fundamental insight into the mechanical properties and cell shape contributions of individual stress fibers and confirming that the ECM is effectively a physical extension of the cell and cytoskeleton, the technologies described here offer a novel approach to spatially map the cytoskeletal mechanics of living cells on the nanoscale.     

The extracellular matrix dimension of skeletal muscle development

Cells anchor to substrates by binding to extracellular matrix (ECM). In addition to this anchoring function however, cell–ECM binding is a mechanism for cells to sense their surroundings and to communicate and coordinate behaviour amongst themselves. Several ECM molecules and their receptors play essential roles in muscle development and maintenance. Defects in these proteins are responsible for some of the most severe muscle dystrophies at every stage of life from neonates to adults. However, recent studies have also revealed a role of cell–ECM interactions at much earlier stages of development as skeletal muscle forms. Here we review which ECM molecules are present during the early phases of myogenesis, how myogenic cells interact with the ECM that surrounds them and the potential consequences of those interactions. We conclude that cell–ECM interactions play significant roles during all stages of skeletal muscle development in the embryo and suggest that this “extracellular matrix dimension” should be added to our conceptual network of factors contributing to skeletal myogenesis.     

Effects of pressure on equatorial x-ray fiber diffraction from skeletal muscle fibers.

When skeletal muscle fibers are subjected to a hydrostatic pressure of 10 MPa (100 atmospheres), reversible changes in tension occur. Passive tension from relaxed muscle is unaffected, rigor tension rises, and active tension falls. The effects of pressure on muscle structure are unknown: therefore a pressure-resistant cell for x-ray diffraction has been built, and this paper reports the first study of the low-angle equatorial patterns of pressurized relaxed, rigor, and active muscle fibers, with direct comparisons from the same chemically skinned rabbit psoas muscle fibers at 0.1 and 10 MPa. Relaxed and rigor fibers show little change in the intensity of the equatorial reflections when pressurized to 10 MPa, but there is a small, reversible expansion of the lattice of 0.7 and 0.4%, respectively. This shows that the order and stability of the myofilament lattice is undisturbed by this pressure. The rise in rigor tension under pressure is thus probably due to axial shortening of one or more components of the sarcomere. Initial results from active fibers at 0.1 MPa show that when phosphate is added the lattice spacing and equatorial intensities change toward their relaxed values. This indicates cross-bridge detachment, as expected from the reduction in tension that phosphate induces. 10 MPa in the presence of phosphate at 11 degrees C causes tension to fall by a further 12%, but not change is detected in the relative intensity of the reflections, only a small increase in lattice spacing. Thus pressure appears to increase the proportion of attached cross-bridges in a low-force state.     

Effect of viscosity on mechanics of single, skinned fibers from rabbit psoas muscle.

Muscle contraction is highly dynamic and thus may be influenced by viscosity of the medium surrounding the myofilaments. Single, skinned fibers from rabbit psoas muscle were used to test this hypothesis. Viscosity within the myofilament lattice was increased by adding to solutions low molecular weight sugars (disaccharides sucrose or maltose or monosaccharides glucose or fructose). At maximal Ca2+ activation, isometric force (Fi) was inhibited at the highest solute concentrations studied, but this inhibition was not directly related to viscosity. Solutes readily permeated the filament lattice, as fiber diameter was unaffected by added solutes (except for an increased diameter with Fi < 30% of control). In contrast, there was a linear dependence upon 1/viscosity for both unloaded shortening velocity and also the kinetics of isometric tension redevelopment; these effects were unrelated to either variation in solution osmolarity or inhibition of force. All effects of added solute were reversible. Inhibition of both isometric as well as isotonic kinetics demonstrates that viscous resistance to filament sliding was not the predominant factor affected by viscosity. This was corroborated by measurements in relaxed fibers, which showed no significant change in the strain-rate dependence of elastic modulus when viscosity was increased more than twofold. Our results implicate cross-bridge diffusion as a significant limiting factor in cross-bridge kinetics and, more generally, demonstrate that viscosity is a useful probe of actomyosin dynamics.     

Diversity of extracellular matrix morphology in vertebrate skeletal muscle

Existing data suggest the extracellular matrix (ECM) of vertebrate skeletal muscle consists of several morphologically distinct layers: an endomysium, perimysium, and epimysium surrounding muscle fibers, fascicles, and whole muscles, respectively. These ECM layers are hypothesized to serve important functional roles within muscle, influencing passive mechanics, providing avenues for force transmission, and influencing dynamic shape changes during contraction. The morphology of the skeletal muscle ECM is well described in mammals and birds; however, ECM morphology in other vertebrate groups including amphibians, fish, and reptiles remains largely unexamined. It remains unclear whether a multilayered ECM is a common feature of vertebrate skeletal muscle, and whether functional roles attributed to the ECM should be considered in mechanical analyses of non-mammalian and non-avian muscle. To explore the prevalence of a multilayered ECM, we used a cell maceration and scanning electron microscopy technique to visualize the organization of ECM collagen in muscle from six vertebrates: bullfrogs (Lithobates catesbeianus), turkeys (Meleagris gallopavo), alligators (Alligator mississippiensis), cane toads (Rhinella marina), laboratory mice (Mus musculus), and carp (Cyprinus carpio). All muscles studied contained a collagen-reinforced ECM with multiple morphologically distinct layers. An endomysium surrounding muscle fibers was apparent in all samples. A perimysium surrounding groups of muscle fibers was apparent in all but carp epaxial muscle; a muscle anatomically, functionally, and phylogenetically distinct from the others studied. An epimysium was apparent in all samples taken at the muscle periphery. These findings show that a multilayered ECM is a common feature of vertebrate muscle and suggest that a functionally relevant ECM should be considered in mechanical models of vertebrate muscle generally. It remains unclear whether cross-species variations in ECM architecture are the result of phylogenetic, anatomical, or functional differences, but understanding the influence of such variation on muscle mechanics may prove a fruitful area for future research.     

Crimped fiber composites: mechanics of a finite-length crimped fiber embedded in a soft matrix

Biomechanics and Modeling in Mechanobiology - Composites comprising crimped fibers of finite length embedded in a soft matrix have the potential to mimic the strain-hardening behavior of tissues...     

Isolation of the heparan sulfate proteoglycans from the extracellular matrix of rat skeletal muscle

We have previously shown that asymmetric collagen-tailed acetylcholinesterase (AChE) is anchored to the extracellular matrix (ECM) by heparan sulfate proteoglycans (HSPGs). Here we present our studies on the characterization of such PGs from the ECM of rat skeletal muscles. After radiolabeling with 35SO4 for 24h, PGs were extracted from the muscle ECM with 4.0 M guanidine-HCl containing protease inhibitors. PGs were subsequently isolated using sequential DEAE-Sephacel chromatography, digestion with chondroitinase ABC, and Sepharose CL-4B. Two different hydrodynamic size species of HSPGs were found. One type had a Mr of 4-6 X 10(5) (Kav = 0.25) as estimated by gel chromatography in the presence of 1% SDS and accounted for 75% of the total HSPGs. The other HSPG had a Mr 1.5-2.5 X 10(5) (Kav = 0.41). The glycosaminoglycan (GAG) side chains (Mr 20,000 and 12,000) were found composed only of heparan sulfate as determined by nitrous acid oxidation and heparitinase treatment. The large-sized HSPG, which is concentrated in synaptic regions, contains only GAG chains of Mr 20,000, suggesting that each HSPG contains only one kind of heparan sulfate chain in its structure. Our results definitively establish by biochemical criteria that the basement membrane of mammalian skeletal muscle contains HSPGs, the likely matrix receptor for the immobilization of the asymmetric collagen-tailed AChE at the neuromuscular junction.     

Lung tissue mechanics and extracellular matrix composition in a murine model of silicosis.

The dynamic mechanical properties of lung tissue and its contents of collagen and elastic fibers were studied in strips prepared from mice instilled intratracheally with saline (C) or silica [15 (S15) and 30 days (S30) after instillation]. Resistance, elastance, and hysteresivity were studied during oscillations at different frequencies on S15 and S30. Elastance increased from C to silica groups but was similar between S15 and S30. Resistance was augmented from C to S15 and S30 and was greater in S30 than in S15 at higher frequencies. Hysteresivity was higher in S30 than in C and S15. Silica groups presented a greater amount of collagen than did C. Elastic fiber content increased progressively along time. This increment was related to the higher amount of oxytalan fibers at 15 and 30 days, whereas elaunin and fully developed elastic fibers were augmented only at 30 days. Silicosis led not only to pulmonary fibrosis but also to fibroelastosis, thus assigning a major role to the elastic system in the silicotic lung.     

A novel proline-rich glycoprotein associated with the extracellular matrix of vascular bundles of Brassica petioles

A panel of monoclonal antibodies (MAC204, MAC236, MAC265) which recognise extracellular matrix glycoproteins implicated in plant-microbe interactions has been used to study glycoprotein antigens in petioles of turnip (Brassica campestris L.). While MAC204 recognised two glycoproteins (gp120 and gp45) with apparent M_r 120 000 and 45 000 in petiole extracts made with 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) buffer containing sodium dodecyl sulfate, MAC236 recognised gp120 but not gp45, and MAC265 gave no or only weak reactivity. Tissue dissection studies established that gp120 was predominantly associated with the vascular bundle whereas gp45 was largely associated with the pith. This was consistent with results from tissue prints probed with MAC204 and MAC236 which also suggested a vascular localisation for gp120. Immunoelectronmicroscopy showed that MAC204 and MAC236 both labelled three-way junctions between cells of the phloem and sclerid fibres. Both gp120 and gp45 were shown to carry epitopes in common with known hy`droxyproline-rich glycoproteins. Unlike gp45, gp120 could be extracted from petioles with Tris buffer alone and then isolated from this extract by trichloroacetic acid treatment (which left gp120 soluble), followed by size-exclusion and ion-exchange chromatography. Amino acid analysis revealed gp120 to be a novel glycoprotein, particularly rich in proline, lysine, valine and threonine but relatively poor in hydroxyproline. The most abundant sugars were arabinose and galactose. The potential role of this very basic cell surface glycoprotein in plant defence against microbes is discussed. Received: 25 November 1996 / Accepted: 12 December 1996     

Sarcomere strain and heterogeneity correlate with injury to frog skeletal muscle fiber bundles.

Sarcomere length and first-order diffraction line width were measured by laser diffraction during elongation of activated frog tibialis anterior muscle fiber bundles (i.e., eccentric contraction) at nominal fiber strains of 10, 25, or 35% (n = 18) for 10 successive contractions. Tetanic tension, measured just before each eccentric contraction, differed significantly among strain groups and changed dramatically during the 10-contraction treatment (P < 0.01). Average maximum tetanic tension for the three groups measured before any treatment was 203.7 +/- 6.8 kN/m2, but after the 10-eccentric contraction sequence decreased to 180.3 +/- 3.8, 125.1 +/- 7.8, and 78.3 +/- 5.1 kN/m2 for the 10, 25, and 35% strain groups, respectively (P < 0.0001). Addition of 10 mM caffeine to the bathing medium decreased the loss of tetanic tension in the 10% strain group but had only a minimal effect on either the 25 or 35% strain groups. Diffraction pattern line width, a measure of sarcomere length heterogeneity, increased significantly with muscle activation and then continued to increase with successive stretches of the activated muscle. Line width increase after each stretch was significantly correlated with the lower yield tension of the successive contractile record. These data demonstrate a direct association and, perhaps, a causal relationship between sarcomere strain and fiber bundle injury. They also demonstrate that muscle injury is accompanied by a progressive increase in sarcomere length heterogeneity, yielding lower yield tension as injury progresses.     

Effect of long-term muscle paralysis on human single fiber mechanics.

This study compared human muscles following long-term reduced neuromuscular activity to those with normal functioning regarding single fiber properties. Biopsies were obtained from the vastus lateralis of 5 individuals with chronic (>3 yr) spinal cord injury (SCI) and 10 able-bodied controls (CTRL). Chemically skinned fibers were tested for active and passive mechanical characteristics and subsequently classified according to myosin heavy chain (MHC) content. SCI individuals had smaller proportions of type I (11 +/- 7 vs. 34 +/- 5%) and IIa fibers (11 +/- 6 vs. 31 +/- 5%), whereas type IIx fibers were more frequent (40 +/- 13 vs. 7 +/- 3%) compared with CTRL subjects (P < 0.05). Cross-sectional area and peak force were similar in both groups for all fiber types. Unloaded shortening velocity of fibers from paralyzed muscles was higher in type IIa, IIa/IIx, and IIx fibers (26, 65, and 47%, respectively; P < 0.01). Consequently, absolute peak power was greater in type IIa (46%; P < 0.05) and IIa/IIx fibers (118%; P < 0.01) of the SCI group, whereas normalized peak power was higher in type IIa/IIx fibers (71%; P < 0.001). Ca(2+) sensitivity and passive fiber characteristics were not different between the two groups in any fiber type. Composite values (average value across all fibers analyzed within each study participant) showed similar results for cross-sectional area and peak force, whereas maximal contraction velocity and fiber power were more than 100% greater in SCI individuals. These data illustrate that contractile performance is preserved or even higher in the remaining fibers of human muscles following reduced neuromuscular activity.     

Alterations induced by cyclosporine A in myocardial fibers and extracellular matrix in rat

Cyclosporine A (CsA) is the first choice immunosuppressant universally used in allotransplantation. However, it has been demonstrated that this drug produces unwanted side effects in several organs and in particular in the kidney and in the heart. While the cardiac toxicity, due to alteration of myocardial prostanoid has been reported, no data are available about the effects of CsA on myocardial cytoarchitecture. We studied the CsA induced alterations of the myocardial structure and of the extracellular matrix components (ECM). To test the ECM enzymatic chances we studied a family of enzymes (matrix metalloproteinase-MMP), responsible for the degradation of extracellular matrix components. In particular we investigated MMPI, MMP2 and MMP9. The study was carried out on two groups of Wistar rats. The group I animals served as a control and were injected subcutaneously daily with castor oil for 21 days. Group II: animals were subcutaneously injected daily with CsA (dose: 15 mg/Kg in castor oil) for 21 days. The group I animals (control) had normal heart architecture and low levels of MMPI, MMP2 and MMP9. The group II animals showed degenerative changes with myocardial fibrosis, low levels of MMP1 and MMP9 but a clear increase in MMP2. We suggest that the myocardial fibrosis was a consequence of the cardiotoxic effect of CsA determining the alteration of the balance between synthesis and degradation of ECM. The increase in MMP2 suggests that this enzyme could play a protective role during myocardial damage and represent a compensatory mechanism for the excessive accumulation of collagen.