A mathematical model of force transmission from intrafascicularly terminating muscle fibers

Many long skeletal muscles are comprised of fibers that terminate intrafascicularly. Force from terminating fibers can be transmitted through shear within the endomysium that surrounds fibers or through tension within the endomysium that extends from fibers to the tendon; however, it is unclear which pathway dominates in force transmission from terminating fibers. The purpose of this work was to develop mathematical models to (i) compare the efficacy of lateral (through shear) and longitudinal (through tension) force transmission in intrafascicularly terminating fibers, and (ii) determine how force transmission is affected by variations in the structure and properties of fibers and the endomysium. The models demonstrated that even though the amount of force that can be transmitted from an intrafascicularly terminating fiber is dependent on fiber resting length (the unstretched length at which passive stress is zero), endomysium shear modulus, and fiber volume fraction (the fraction of the muscle cross-sectional area that is occupied by fibers), fibers that have values of resting length, shear modulus, and volume fraction within physiologic ranges can transmit nearly all of their peak isometric force laterally through shearing of the endomysium. By contrast, the models predicted only limited force transmission ability through tension within the endomysium that extends from the fiber to the tendon. Moreover, when fiber volume fraction decreases to unhealthy ranges (less than 50%), the force-transmitting potential of terminating fibers through shearing of the endomysium decreases significantly. The models presented here support the hypothesis that lateral force transmission through shearing of the endomysium is an effective mode of force transmission in terminating fibers.     

Localization of alpha 7 integrins and dystrophin suggests potential for both lateral and longitudinal transmission of tension in large mammalian muscles

Non-primate mammalian muscles with fascicles above 35 mm in length are composed predominantly of arrays of short, non-spanning muscle fibres, which terminate within the belly of the muscle fascicle at one or both ends. We have previously described the morphological form of various muscle-to-muscle and muscle-to-matrix junctions which are likely involved in tension transmission within one such muscle - the guinea pig sternomastoid muscle (Young et al. 2000). Here, we use immunohistochemistry to investigate the cell adhesion molecules present at these junctions. We find strong immunoreactivity against the alpha 7B integrin subunit and dystrophin, and slight reactivity against the alpha 7A integrin at all intrafascicular fibre terminations (IFTs), as well as at the muscle-tendon junction (MTJ). Tenascin, the sole ligand for alpha 9 beta 1 integrin, was absent from IFTs but present at the MTJ, suggesting the two sites are molecularly distinct. In addition to their expression at junctional sites, alpha 7B integrin and dystrophin were also expressed ubiquitously along the non-junctional sarcolemma, suggesting potential involvement in diffuse lateral transmission of tension between adjacent fibres. We conclude that the distribution of alpha 7 beta 1 integrins and dystrophin in series-fibred muscles suggests they are involved in transmission of tension from intrafascicularly terminating fibres to neighbouring fibres lying both in-series and in-parallel, via the extracellular matrix (ECM).     

Injury to nerves and the initiation of amphibian limb regeneration

The force produced within skeletal muscle fibers is transmitted to the bone via a myotendinous junction. This junctional region was examined by light and electron microscopy in the sartorius muscles of three Rana temporaria. The muscle fibers tapered and inserted at an angle of about 25 degrees with the connective tissue fascia near the bone. The composition of the structures within the last 100 microns of the fiber was analyzed morphometrically. The T-system, terminal cisternae, and caveolae were the same as in the central region of the muscle fiber. However, the mitochondrial content was higher and the volume of longitudinal sarcoplasmic reticulum was lower than elsewhere in the fiber. The membrane at the end of the fiber had extensive villiform processes interdigitating with the tendon. The surface area of the membrane around the villiform processes was estimated with point-counting techniques and calculated from the stereological equations appropriate for partially anisotropic structures. The extra membrane involved in the myotendinous junction was about 32 times that of the cross-sectional area of the fiber. Part of this additional membrane contained specialized adherens junctions through which the contractile proteins of the muscle are anchored to collagen. The increased area at the myotendinous junction presumably provides greater mechanical strength than a flat termination. The high values of membrane capacitance and specific resistance measured electrophysiologically at the end of the fiber also can be attributed to the characteristics of the terminal membrane structure.     

In-series fiber architecture in long human muscles

The fiber architecture of adult human sartorius and gracilis muscles was examined using a combination of fiber microdissections and histological methods. Intact fibers were dissected from fascicles of muscle strips that were digested in nitric acid. All of these fibers terminate intrafascicularly by tapering to a fine strand at one or both ends. They measure 4–20 cm after correction for shrinkage. Systematic dissections of 1 cm long blocks sampled at intervals along the muscle length suggest that tapered fiber endings occur at all locations along the muscle but are most common centrally; here they accounted for up to 14% of dissected fibers in each block. Transverse sections of muscle confirm that fiber profiles with small diameters occur at all levels of the muscle but are especially common in sections more than 5 cm from its origin or insertion. The architectural arrangement demonstrated here suggests that long human muscles, like muscles in other species, are composed of relatively short, in-series fibers. This has many implications for the neural activation and force-developing behavior of these muscles that must be considered when paralyzed muscles are reanimated using electrical stimulation. Further, it may predispose long muscles to certain types of neuromuscular damage and dysfunction. © 1993 Wiley-Liss, Inc.     

The fine structure of myotendinous and myo-epithelial junctions in the guinea pig tongue (author's transl)]

The insertion of muscle fibers in the subepithelial connective tissue layer of the guinea pig tongue was studied light and electron microscopically. Fibers of the tractus verticalis approach the epithelium penetrating the lamina propria, both the reticular and papillar layer. Terminating muscle fibers split up and form branching finger-like cytoplasmic processes. The myotendinous junctions of such terminal processes fine structurally correspond to myotendinous junctions generally observed in skeletal or smooth muscles. The entire brush-like formation, however, is more far-reaching and highly differentiated. Filament bundles (spine-like profiles) originate from the plasmalemma and extend to the lamina densa of the basal lamina, especially in those regions where actin filaments are attached to the plasmalemma. Microfibrils (10 to 12 nm diameter) reach the lamina densa of the basal lamina. They form bundles which are continuous with fibrotubular strands of elaunin fibers and elastic fiber microfibrils. Furthermore, microfibrils are interwoven with collagen fibrils.     

SNAREs in native plasma membranes are active and readily form core complexes with endogenous and exogenous SNAREs

Skeletal muscles display a remarkable diversity in their arrangement of fibers into fascicles and in their patterns of innervation, depending on functional requirements and species differences. Most human muscle fascicles, despite their great length, consist of fibers that extend continuously from one tendon to the other with a single nerve endplate band. Other mammalian muscles have multiple endplate bands and fibers that do not insert into both tendons but terminate intrafascicularly. We investigated whether these alternate structural features may dictate different modes of cell hypertrophy in two mouse gracilis muscles, in response to expression of a muscle-specific insulin-like growth factor (IGF)-1 transgene (mIGF-1) or to chronic exercise. Both hypertrophic stimuli independently activated GATA-2 expression and increased muscle cross-sectional area in both muscle types, with additive effects in exercising myosin light chain/mIGF transgenic mice, but without increasing fiber number. In singly innervated gracilis posterior muscle, hypertrophy was characterized by a greater average diameter of individual fibers, and centralized nuclei. In contrast, hypertrophic gracilis anterior muscle, which is multiply innervated, contained longer muscle fibers, with no increase in average diameter, or in centralized nuclei. Different modes of muscle hypertrophy in domestic and laboratory animals have important implications for building appropriate models of human neuromuscular disease.     

Alpha-actinin is absent from the terminal segments of myofibrils and from subsarcolemmal densities in frog skeletal muscle

The presence and distribution of alpha-actinin, an actin-bundling protein, was investigated at sites where frog skeletal muscle forms junctions with tendon collagen fibers. These sites, called myotendinous junctions, are regions where myofibrils terminate and where the force of muscular contraction is transmitted from muscle cells to the substratum. An antibody manufactured to chicken smooth muscle alpha-actinin was used as a probe for alpha-actinin localization in this study. The cross-reactivity of this antibody with frog skeletal muscle alpha-actinin is demonstrated in immunoblots of one-dimensional (1D) electrophoretic separations of muscle proteins. Immunofluorescent localization of anti-alpha-actinin and electron microscopic immunolabelling confirms that the antibody binds to Z-discs with high affinity. However, in sections treated for electron microscopy with affinity-purified anti-alpha-actinin and a ferritin-conjugated, second antibody, there was no significant difference between experimental or control preparations in the number of ferritin grains overlying dense, subsarcolemmal material at junctional or non-junctional regions. Furthermore, Z-discs near myotendinous junctions displayed less binding of anti-alpha-actinin than Z-discs located several micrometers or more from the cells' termini. These findings indicate that thin filaments are not bundled by alpha-actinin near the sarcolemma. The results also provide evidence for molecular heterogeneity between Z-discs at the ends of muscle cells compared with other regions of the cell in that the terminal Z-discs of myofibrils contain very little or no alpha-actinin relative to non-terminal Z-discs.     

Extracellular matrix and transmembrane linkages at the termination of intrafusal fibers and the outer capsule in chicken muscle spindles

Attachments of intrafusal fibers and of the outer spindle capsule at the far polar region were examined by immunohistochemistry in serially sectioned chicken leg muscles. Patterns of distribution of connective tissues and intracellular filaments suggest that, in this segment of the muscle spindle, intrafusal fibers bind laterally with the capsule. Contrary to extrafusal fibers at myotendinous junctions, folded plasmalemmas at the ends of intrafusal fibers were rare. Thus, there was little end-to-end interlocking between intrafusal fibers and the extracellular matrix. The tapered contours of terminating intrafusal fibers resembled those of extrafusal fibers which end in fascicles without tendinous connections. At points where the distal portions of intrafusal fibers closely adjoined and overlapped extrafusal fibers, α-actinin, vinculin, filamin, talin, β₁ integrin, spectrin, and dystrophin occurred with moderate to great frequency. It is generally accepted that these compounds are links in molecular chains that extend from the intracellular space across cell membranes to the extracellular matrix. Their location along substantial lengths of extrafusal fibers, distal capsule, and terminating intrafusal fibers suggests the presence of numerous transverse connections between elements of the terminal portion of the spindle and nonspindle tissues. Hence, it is likely that forces monitored by chicken spindles in muscles undergoing length changes are transferred from extrafusal fibers and extracellular matrix to the receptors in large part via lateral shear instead of by longitudinal tension. © 1996 Wiley-Liss, Inc.     

Vinculin in subsarcolemmal densities in chicken skeletal muscle: localization and relationship to intracellular and extracellular structures

Using immunocytochemical methods we have studied the distribution of vinculin in the anterior and posterior latissimus dorsi skeletal (ALD and PLD, respectively) muscles of the adult chicken. The ALD muscle is made up of both tonic (85%) and twitch (15%) myofibers, and the PLD muscle is made up entirely of twitch myofibers. In indirect immunofluorescence, antivinculin antibodies stained specific regions adjacent to the sarcolemma of the ALD and PLD muscles. In the central and myotendinous regions of the ALD, staining of the tonic fibers was intense all around the fiber periphery. Staining of the twitch fibers of both ALD and PLD muscles was intense only at neuromuscular junctions and myotendinous regions. Electron microscopy revealed subsarcolemmal, electron-dense plaques associated with the membrane only in those regions where vinculin was localized by immunofluorescence. Using antivinculin antibody and protein A conjugated to colloidal gold, we found that the electron-dense subsarcolemmal densities in the tonic fibers of the ALD contain vinculin; no other structures were labeled. The basal lamina overlying the densities appeared to be connected to the sarcolemma by fine, filamentous structures, more enriched at these sites than elsewhere along the muscle fiber. Increased amounts of endomysial connective tissue were often found just outside the basal lamina near the densities. In tonic ALD muscle fibers, the subsarcolemmal densities were present preferentially over the I-bands. In partially contracted ALD muscle, subsarcolemmal densities adjacent to the Z-disk appeared to be connected to that structure by short filaments. We propose that in the ALD muscle, through their association with the extracellular matrix, the densities stabilize the muscle membrane and perhaps assist in force transmission.     

Adhesive strength of single muscle cells to basement membrane at myotendinous junctions

Whole muscles loaded to failure frequently fail at or near myotendinous junctions. The present investigation was directed toward determining the breaking stress and failure site of intact and injured myotendinous junction preparations consisting of muscle cells dissected free from surrounding parallel structures but still attached to tendon collagen fibers. These tests show that the breaking stress for intact myotendinous units is 2.7 x 10(5) N/m2, expressed relative to cell cross-sectional area. Failure occurs immediately external to the junction membrane between the cell membrane and lamina densa of the basement membrane. Site and stress at failure are independent of strain and strain rate over a biologically relevant range. Breaking stress in the plane of the membrane, corrected for membrane folding, is 1.2 X 10(4) N/m2. This value is not significantly greater than stress at maximum isometric tension for these cells at these sarcomere lengths. After compression injury, cells fail within the compression site at significantly lower stress (1.9 X 10(5) N/m2). These findings suggest that, in muscle strain injuries that occur under conditions simulated here, failure occurs at myotendinous junctions unless the muscle has suffered previous compression injury leading to failure within the muscle.     

Motor nerve topology reflects myocyte morphology in hamster retractor and epitrochlearis muscles

Neuromuscular activation is a primary determinant of metabolic demand and oxygen transport. The m. retractor and m. epitrochlearis are model systems for studying metabolic control and oxygen transport; however, the organization of muscle fibers and motor nerves in these muscles is unknown. We tested whether the topology of motor innervation was related to the morphology of muscle fibers in m. retractor and m. epitrochlearis of male hamsters ( approximately 100 g). Respective muscles averaged 47 and 12 mm in length 100 and 35 mg in mass. Staining for acetylcholinesterase revealed neuromuscular junctions arranged in clusters throughout m. retractor and as a central band across m. epitrochlearis, suggesting differences in fiber morphology. For both muscles, complete cross-sections contained approximately 1,700 fibers. Fiber cross-sectional areas were distributed nearly normal in m. epitrochlearis (mean = 1,559 +/- 17 microm(2)) and skewed left (P < 0.05) in m. retractor (mean = 973 +/- 15 microm(2)). Single fiber length (Lf) spanned muscle length (Lm) in m. epitrochlearis, while fibers tapered to terminate within m. retractor (Lf/Lm = 0.43 +/- 0. 02). With myelin staining, a single branch of ulnar nerve projected axons across the midregion of m. epitrochlearis. For m. retractor, the spinal accessory nerve branched to give rise to proximal and distal regions of innervation, with intermingling of axons between nerve branches. Nerve bundle cross-sections stained for acetylcholinesterase indicate that each motor axon projects to an average of 65 muscle fibers in m. epitrochlearis and 100 in m. retractor. Differences in fiber morphology, innervation topology, and neuromuscular organization may contribute to the heterogeneity of metabolic demand and oxygen supply in skeletal muscle.     

Talin at myotendinous junctions

Junctions formed by skeletal muscles where they adhere to tendons, called myotendinous junctions, are sites of tight adhesion and where forces generated by the cell are placed on the substratum. In this regard, myotendinous junctions and focal contacts of fibroblasts in vitro are analogues. Talin is a protein located at focal contacts that may be involved in force transmission from actin filaments to the plasma membrane. This study investigates whether talin is also found at myotendinous junctions. Protein separations on SDS polyacrylamide gels and immunolabeling procedures show that talin is present in skeletal muscle. Immunofluorescence microscopy using anti-talin indicates that talin is found concentrated at myotendinous junctions and in lesser amounts in periodic bands over nonjunctional regions. Electron microscopic immunolabeling shows talin is a component of the digitlike processes of muscle cells that extend into tendons at myotendinous junctions. These findings indicate that there may be similarities in the molecular composition of focal contacts and myotendinous junctions in addition to functional analogies.     

Desmin at myotendinous junctions

Myofibrils are linked to the cell membrane at myotendinous junctions located at the ends of muscle fibers, and at costameres, sites positioned periodically along lateral surfaces of muscle cells. Both of these sites are enriched in proteins that link active components of myofibrils to the cell membrane. Costameres are also enriched in desmin intermediate filaments that link passive components of myofibrils to the lateral surfaces of muscle cells. In this study, the possibility that desmin is also found between the terminal Z-disk of myofibrils and the myotendinous junction membrane is examined by immunocytochemistry and by KI-extraction procedures. Data presented show that desmin is located in the filamentous core of cellular processes at myotendinous junctions at sites 30 nm or more from the membrane. This core lies deep to subsarcolemmal material previously shown to contain talin, vinculin, and dystrophin. The distance from desmin to the membrane suggests desmin does not interact directly with membrane proteins at the junction. Immunoblots and indirect immunofluorescence of junctional regions of muscle compared to nonjunctional regions show no apparent enrichment of desmin at junctional sites, although vinculin, another costameric and junctional component, is significantly enriched at junctional regions. These findings show that passive elements of myofibrils may be continuous from myotendinous junctions of muscle origin to insertion via desmin filaments located between terminal Z-disks and the junctional membrane. This can provide a system in parallel to that involving thin filaments, vinculin, and talin for linking myofibrils to the cell membrane at myotendinous junctions.     

Formation of myoneural and myotendinous junctions in the chick embryo

Summary The mode of formation of the myoneural and myotendinous junctions was investigated in the thigh muscles of the chick embryo. Myotendinous junctions first appeared on day 11 of incubation, whereas myoneural junctions developed on day 12. Intracellular AChE activity in the muscles increased by the 12th day of incubation, and decreased rapidly after the formation of the myoneural junctions. Light and electron microscopically, AChE activity was demonstrated in the nuclear envelope, sarcoplasmic reticulum, Golgi complex, and in large granules which appeared to be derived from the Golgi complex. Large granules showing an intense AChE activity accumulated in the sarcoplasm at the poles of the muscle fiber before the formation of myotendinous junctions. After the translocation of this intracellular enzyme onto the sarcolemma, most likely the result of an exocytosis of the granules, the myotendinous junctions were formed. The AChE-rich granules present in the middle of myotubes developed into spindle- or comma-shaped cisternae which were located in the sarcoplasm just below the presumptive motor endplates. The present results suggest that the transport of AChE-rich granules to the sarcolemma is the first step in the formation of myoneural and myotendinous junctions.This work was carried out under grant 38848 from the Ministry of Education of Japan     

Early innervation of skeletal muscle during tail regeneration in urodele amphibians.

The innervation pattern of skeletal muscles was studied in the normal and regenerating tail of Notophthalmus viridescens. Silver staining for nerve endings and histochemical localization of acetylcholinesterase (AChE) were used for light microscopy. In In normal musculature, AChE positive reactions were localized at the ends of the muscle fibers where they are anchored on connective tissue septa by myotendinous junctions. At this level, silver staining shows nerve terminals forming endplates. During regeneration, positive reactions for AChE appear de novo as dense plates localized at the ends of the newly formed myotubes. The mechanisms involved in the localization of AChE on this surface seem to operate before previous local contacts by nerve terminals. From the ultrastructural data and immunohistochemical results with anti-laminin antibody, these observations suggest that regenerating muscle fibers determine a region of post-synaptic specialization in close relation with the organization of myotendinous regions and basement membrane formation. Nerve-muscle contacts appear at these levels at stage IV (15-20 days after amputation) in the stump and in the rostral part of the regenerate (transition zone). These nerve terminals are provided by the disorganized peripheral nervous system of the injured segment. In the regenerate a similar pattern of AChE reaction can be seen in every myotube, differentiating according to a rostro-caudal gradient. Innervation at the ends of the muscle fibers is in spatiotemporal relation with the exists of the ventral roots from the regenerating nerve cord as the regenerate continues to grow in length.     

Fiber architecture of canine abdominal muscles.

During respiration, abdominal muscles experience loads, not only in the muscle-fiber direction but also transverse to the fibers. We wondered whether the abdominal muscles exhibit a fiber architecture that is similar to the diaphragm muscle, and, therefore, we chose two adjacent muscles: the internal oblique (IO), with about the same muscle length as the diaphragm, and the transverse abdominis (TA), which is twice as long as the diaphragm. First, we used acetylcholinesterase staining to examine the distribution of neuromuscular junctions on both surfaces of the TA and IO muscles in six dogs. A maximum of four irregular bands of neuromuscular junctions crossed the IO, and as many as six bands crossed the TA, which is consistent with a discontinuous fiber architecture. In six additional dogs, we examined fiber architecture of these muscles by microdissecting 103 fascicles from the IO and 139 from the TA. Each fascicle contained between 20 and 30 muscle fibers. The mean length of nonspanning fibers (NSF) ranged from 2.8 +/- 0.3 cm in the IO to 4.3 +/- 0.5 cm in the TA, and the mean length of spanning fibers ranged from 4.3 +/- 0.5 cm in the IO to 7.6 +/- 1.4 cm in the TA. NSF accounted for 89.6 +/- 1.5% of all fibers dissected from the IO and 99.1 +/- 0.2% of all fibers dissected from the TA. The percentage of NSF with both ends tapered was 6.2 +/- 1.0 and 41.0 +/- 2.3% for IO and TA, respectively. These data show that fiber architecture in either IO or TA is discontinuous, with much more short-tapered fibers in the TA than in the IO. When abdominal muscles are submaximally activated, as during both normal expiration and maximal expiratory efforts, muscle force could be transmitted to the cell membrane and to the extracellular intramuscular connective tissue by shear linkage, presumably via structural transmembrane proteins.     

Increase in rat soleus myotendinous interface after a 14-d spaceflight

Myotenclinous junctions (MTJs) transmit contractile force from skeletal muscles to tendons. The effects of a 14-d spaceflight on MTJ were studied in the soleus muscle of male adult Sprague Dawley rats by transmission electron microscopy and histomorphometric techniques. We showed that the length of the junctional membrane relative to the muscle fiber diameter increased by 58% after 14 d of spaceflight. This increase accompanies morphological changes at MTJs. The flight MTJs appeared more shredded. The ends of the muscle fibers exhibited T tubule dilatation, swollen mitochondria, Z-disk streaming, loss of myofilaments, a thinning down of subplasmalemmal densities, multivesicular bodies and signs of junctional membrane and basal lamina remodelling. The ultrastructural observations suggest that the increase in myotendinous interface could result from the extracellular matrix spreading into remodelling muscle fiber, whereas the constraints related to unloading were reduced by spaceflight conditions.     

Myotendinous junctions of tonic muscle cells: structure and loading

Summary Regions within frog semitendinosus muscle that are rich in tonic muscle cells were identified histochemically by myosin adenosine triphosphatase- and succinic dehydrogenase-staining procedures. Bundles of cells still attached to tendinous insertions were removed from those sites, prepared for electron microscopy and sectioned longitudinally through their myotendinous junctions. Tonic cells were identified by electron-microscopic criteria and their myotendinous junctions' morphology evaluated by morphometry. Although junctional components appear identical to those in twitch cells, the degree of membrane folding increases tonic junction area by a factor of 50.2 whereas twitch cells' junctional area is increased 22.2 times by folding relative to cells terminating as right circular cylinders. Calculations show that the tonic cell junction bears average loads of 3.4×10³ N · m^-2 during maximum force generation and that nearly all of the load is borne as shear stress at the junction. The junctions of twitch cells bear average loads of 1.6×10⁴ N · m^-2 during peak tension. The findings indicate that the magnitude of loading does not alone determine the degree of junctional membrane folding. Interpretation of the data in view of viscoelastic behavior of membranes indicates that duration of loading may be a functionally important correlate to degree of membrane folding at myotendinous junctions.     

The nervous environment of individual smooth muscle cells of the guinea pig vas deferens

Smooth muscle cells of the external longitudinal coat of the guinea pig vas deferens were followed for 480 mu at 4.5-mu intervals. Muscle bundles and fibers interwove, facilitating intermuscular and neuromuscular contacts. The ribbon- or rodlike muscle cells were about 450 mu long, 3,000 mu3 in volume, and 4,500 mu2 in area. The thickened nuclear zone day anywhere along the middle one-third of the cell. Intercellular distances were 500-800 A. Intrusions were rare, and tight-junctions absent. At any level in a field of 80 muscle fibers there were 10-15 nerve bundles, each containing several varicose axons. Bundles and axons divided. Axons, en passage, were frequently within 500-1,000 A of a muscle fiber. En passage close contacts were rate. Axon terminations were bare, and bare axons invariably terminated. Bare terminations had scattered vesicle-laden varicosities and were from 10-60 mu in length, and all ended within 500 A of muscle fibers. Some made close contact with muscle fibers. Less than half of the muscle cells received this close contact, but some cells were approached by more than one termination. Most terminations involved more than one cell. Some cells had little or no innervation. Some groups of cells had a rich innervation. There was very little evidence of sensory innervation. These conclusions are not valid for other smooth muscles.