Nonlinear stiffness--force relationships in whole mammalian skeletal muscles

Small, sinusoidal length changes were superimposed on isometric contractions of fast- and slow-twitch mouse muscles, which were stimulated maximally via their nerves. Stiffness increased with increasing frequency of sinusoidal stimulation, but the relative time course of force and stiffness changes during twitch, tetanic, or partially fused contractions was quite invariant over a range of frequencies in both muscles. Typically, stiffness increases more rapidly than force during contraction and decreases less rapidly during relaxation. This pattern was observed at various temperatures and with various numbers of stimuli. It can be described by a nonlinear relation between stiffness and force with some hysteresis. The presence in the muscle of parallel and series elastic elements, whose stiffness varies with force, may account for the nonlinear relation. This nonlinearity can be used to relate the patterns for summation of force and stiffness observed with brief trains of stimuli under a variety of conditions.     

Trophic regulation of acetylcholinesterase isoenzymes in adult mammalian skeletal muscles

This work addresses the physiological regulation of skeletal muscle acetylcholinesterase (AChE) isoforms by examining endplate-enriched samples from adult rat gracilis muscles 48 h after: lowintensity treadmill exercise; obturator nerve transection; nerve impulse conduction blockade by tetrodotoxin; acetylcholine (ACh) receptor (AChR) inactivation by -bungarotoxin; and, addition of obturator nerve extracts to muscles in organ culture. Results document the important role(s) of functional AChRs and ACh-AChR interactions in the differential control of individual AChE isoenzymes. A theoretical model based on these and other findings considers that: AChR activation by spontaneously released ACh is the only neural factor required for the maintenance of G₁+G₂ AChE; the amount of A₁₂ AChE is determined by the combined effects of ACh and another neurogenic substance; although mechanisms intrinsic to myofibers control normal levels of G₄ AChE, enhanced production of this isoform is initiated through increasing the frequency of ACh-AChR interactions.Special issue dedicated to Dr. Frederick E. Samson     

The origin of secondary myotubes in mammalian skeletal muscles: ultrastructural studies

The distribution of secondary myotubes and undifferentiated mononucleated cells (presumed to be myoblasts) within foetal IVth lumbrical muscles of the rat was analyzed with serial section electron microscopy. In all myotube clusters for which the innervation zone was located, every secondary myotube overlapped the end-plate region of the primary myotube. No secondary myotubes were ever demonstrated to occur at a distance from the primary myotube innervation zone. This indicates that new secondary myotubes begin to form only in the innervation zone of the muscle. Some young secondary myotubes made direct contact with a nerve terminal, but we cannot say if this is true for all developing secondary myotubes. Myoblasts were not clustered near the innervation zone, but were uniformly distributed throughout the muscle. Myoblasts were frequently interposed between a primary and a secondary myotube, in equally close proximity to both cell membranes. We conclude that specificity in myoblast-myotube fusion does not depend on restrictions in the physical distribution of myoblasts within the muscle, and therefore must reflect more subtle mechanisms for intercellular recognition.     

Modulation of caffeine contractures in mammalian skeletal muscles by variation of extracellular potassium

Caffeine contractures were induced after K⁺ -conditioning of skeletal muscles from pigs and mice. K⁺ -conditioning is defined as the partial depolarization caused by increasing external potassium (K) with [K⁺]×[Cl^?] constant. Conditioning depolarizations that rendered muscles refractory to brief electrical stimulation still enhanced the contracture tension elicited by subsequent direct caffeine stimulation of sarcoplasmic reticulum (SR) calcium release. The effects of K⁺ -conditioning on caffeine-induced contractures of intact cell bundles reached a maximum at 15–30 mM K and then progressively declined at higher [K⁺]₀. Conditioning with 30 mM K⁺ for 5 min, which inactivates excitation-contraction (EC) coupling in response to action potentials, both increased the magnitude of caffeine contractures 2–10-fold and shifted the contracture threshold toward lower caffeine concentrations. Enhanced sensitivity to caffeine was inhibited by dantrolene (20 μM) and its watersoluble analogue azumolene (150 μM). These drugs decreased caffeine-induced contractures following depolarization with 4–15 mM K⁺ to 25–50% of control tension. The inorganic anion perchlorate (CIO), which like caffeine potentiates twitches, increased caffeine-induced contractures ～? twofold after K⁺ -conditioning (>4 mM). The results suggest that CIO and dantrolene, in addition to caffeine, also influence SR calcium release either directly or by mechanism(s) subsequent to depolarization of the sarcolemma. Moreover, since CIO is known to shift the voltage-dependence of intramembrane charge movement, CIO may exert effects on the transverse-tubule voltage sensors as well as the SR. © 1995 Wiley-Liss, Inc.     

Passive forces in mammalian skeletal muscle: a freely-jointed and worm-like chain.

The passive mechanical properties of whole muscle in active and nonactive states are compared. The experimental results are presented as stress-strain curves, which are analyzed in the framework of the current theoretical background [viz. the freely-jointed chain model (FJCM) and the worm-like chain model (WLCM)] in a semi-quantitative fashion. This analysis shows that both models can explain the mechanical behavior of whole muscle in non-active state. In the active state, the presence of crossbridges alters the mechanical response, leading to a markedly different behavior, as expected. A discussion of the mechanisms involved and the interpretation of the parameters required for the fitting of the stress-strain curves is also presented.     

Static and dynamic x-ray diffraction recordings from living mammalian and amphibian skeletal muscles

Static and time-resolved two-dimensional x-ray diffraction patterns, recorded from the living mouse diaphragm muscle, were compared with those from living frog sartorius muscle. The resting pattern of mouse muscle was similar to that of frog muscle, and consisted of actin- and myosin-based reflections with spacings basically identical to those of frog. As a notable exception, the sampling pattern of the myosin layer lines (MLL's) indicated that the mouse myofilaments were not organized into a superlattice as in frog. The intensity changes of reflections upon activation were also similar. The MLL's of both muscles were markedly weakened. Stereospecific (rigorlike) actomyosin species were not significantly populated in either muscle, as was evidenced by the 6th actin layer line (ALL), which was substantially enhanced but without a shift in its peak position or a concomitant rise of lower order ALL's. On close examination of the mouse pattern, however, a few lower order ALL's were found to rise, slightly but definitely, at the position expected for stereospecific binding. Their quick rise after the onset of stimulation indicates that this stereospecific complex is generated in the process of normal contraction. However, their rise is still too small to account for the marked enhancement of the 6th ALL, which is better explained by a myosin-induced structural change of actin. Since the forces of the two muscles are comparable regardless of the amount of stereospecific complex, it would be natural to consider that most of the force of skeletal muscle is supported by nonstereospecific actomyosin species.     

Interaction of aldolase with thin filaments within I-disks, isolated from skeletal muscles

Using electron microscopy and optical diffraction, Ca2+-dependent binding of a glycolytic enzyme (aldolase) to thin filaments of isolated skeletal muscle I-disks have been revealed. On the micrographs of negatively stained I-disks the cross-striation determined by troponin-tropomyosin complex distribution has a period of about 38 nm. The width of troponin-tropomyosin stripes is 5-6 nm. On the optical diffraction patterns from isolated I-disks the meridional reflections measuring 38.5, 19.2, 12.8 nm are present. On the micrographs of isolated I-disks, treated with aldolase in the absence of Ca2+ (1 mM EGTA) the width of periodic transverse stripes (period approximately 38 nm) increases from 5-6 nm to 25-28 nm due to the interaction of aldolase with thin filaments. On the optical diffraction patterns from I-disks treated with aldolase in the absence of Ca2+ (1 mM EGTA) the strong meridional reflection equal to 38.5 nm is present, while the reflections equal to 19.2 nm are absent. The optical diffraction patterns from I-disks treated with aldolase in the presence of Ca2+ (greater than or equal to 10(-5) M) do not, as a rule, differ from those obtained from I-disks not treated with aldolase, i.e. they contain the three above reflections. The binding of aldolase to thin filaments in the absence of Ca2+ is the reason of disappearance of meridional reflections equal to 19.2 and 12.8 nm.     

Endothelial cells within embryonic skeletal muscles: a potential source of myogenic progenitors

We investigated whether the vessel-associated or endothelial cells within mouse embryo muscles can be a source of myogenic progenitors. Immunodetection of the stem cell surface markers, CD34 and Flk1, which are known to characterize the endothelial lineage, was done throughout the course of embryo muscle development. Both markers appeared to be restricted to the vessel-associated cells. On the basis of CD34 labeling, the reactive cells were purified by magnetic-bead selection from the limb muscles of 17-dpc desmin+/-LacZ mouse embryos and characterized by fluorescence-activated cell sorting. The cells in the selected CD34(+) population appeared to be approximately 95% positive for Flk1, but usually negative for CD45. We demonstrated that in vitro the CD34(+)/Flk1(+) population differentiated into endothelial cells and skeletal myofibers. When transplanted into mdx mouse muscle, this population displayed a high propensity to disperse within the recipient muscle, fuse with the host myofibers, and restore dystrophin expression. The marked ability of the embryonic muscle endothelial cells to activate myogenic program could be related to their somitic origin.     

Myonuclear birthdates distinguish the origins of primary and secondary myotubes in embryonic mammalian skeletal muscles

Myotubes were isolated from enzymically disaggregated embryonic muscles and examined with light microscopy. Primary myotubes were seen as classic myotubes with chains of central nuclei within a tube of myofilaments, whereas secondary myotubes had a smaller diameter and more widely spaced nuclei. Primary myotubes could also be distinguished from secondary myotubes by their specific reaction with two monoclonal antibodies (MAbs) against adult slow myosin heavy chain (MHC). Myonuclei were birth dated with [3H]thymidine autoradiography or with 2-bromo-5'-deoxyuridine (BrdU) detected with a commercial monoclonal antibody. After a single pulse of label during the 1-2 day period when primary myotubes were forming, some primary myotubes had many myonuclei labelled, usually in adjacent groups, while in others no nuclei were labelled. If a pulse of label was administered after this time labelled myonuclei appeared in most secondary myotubes, while primary myotubes received few new nuclei. Labelled and unlabelled myonuclei were not grouped in the secondary myotubes, but were randomly interspersed. We conclude that primary myotubes form by a nearly synchronous fusion of myoblasts with similar birthdates. In contrast, secondary myotubes form in a progressive fashion, myoblasts with asynchronous birthdates fusing laterally with secondary myotubes at random positions along their length. These later-differentiating myoblasts do not fuse with primary myotubes, despite being closely apposed to their surface. Furthermore, they do not generally fuse with each other, as secondary myotube formation is initiated only in the region of the primary myotube endplate.     

Quantitation and immunocytochemical localization of ubiquitin conjugates within rat red and white skeletal muscles

We employed solid-phase immunochemical methods to probe the dynamics of ubiquitin pools within selected rat skeletal muscles. The total ubiquitin content of red muscles was greater than that of white muscles, even though the fractional conjugation was similar for both types of muscle. The specificity for conjugated ubiquitin in solid-phase applications, previously demonstrated for an affinity-purified antibody against SDS-denatured ubiquitin, was retained when used as a probe for ubiquitin-protein adducts in tissue sections. Immunohistochemical localization revealed that differences in ubiquitin pools derived from the relative content of red (oxidative) vs white (glycolytic) fibers, with the former exhibiting a higher content of ubiquitin conjugates. Subsequent immunogold labeling demonstrated statistically significant enhanced localization of ubiquitin conjugates to the Z-lines in both red and white muscle fiber types.     

Aging of human skeletal muscles

Normal aging in humans is associated with progressive decrease in skeletal muscle mass and strength (sarcopenia) which contributes to frailty and falls. The age associated changes in body composition result from lower levels of anabolic hormones, oxidative damage, neuromuscular alterations and a general decrease in muscle protein turnover. In this review we discuss the potential mechanisms and physical activity as prevention and treatment of sarcopenia.     

Regeneration of entire skeletal muscles

There are several experimental models for producing the regeneration of entire mammalian muscles. The most commonly used are mincing and free muscle grafting. Immediately after both mincing and grafting, the muscle is completely divorced from any connections with the host. To regenerate and become functional, the muscle must become reintegrated with the body of the host. The three major reintegrative phenomena--revascularization, reinnervation, and the reestablishment of tendon connections--are discussed in the context of muscle regeneration. The functional development of regenerating muscle closely resembles the normal ontogenetic pattern. Final functional differentiation of a regenerating muscle depends on the establishment of neuromuscular synapses.     

Glycogen phosphorylase of skeletal muscles

A review is given on the affinity modification of pyridoxal phosphate and AMP-binding sites as well as on the chemical modification of essential amino acid residues of phosphorylase (histidine residue of the substrate-binding site and cysteine residue of the coenzyme-binding site). The role of allosteric effectors (AMP and glucose-6-phosphate) and functionally important centers of the protein in conformational transitions of rabbit muscle phosphorylase b is discussed. The kinetic properties of rabbit and bovine muscle phosphorylase are compared. Bovine muscle phosphorylase is shown to be a partly phosphorylated form of the enzyme. Some peculiarities of the pH-dependence of kinetic behaviour of the hybrid form of the bovine muscle enzyme are discussed.     

Tensile forces in skeletal structures

Recent hypotheses about the mechanisms of adaptation of bone to impressed mechanical forces differ according to the extent to which tensile stresses are thought to be functionally important in bone. A pilot study of three anatomical regions by means of the photoelastic analogy suggests the possibility that net tension rarely exists in significantly large regions of bones during normal function. Thus the examination of two situations (opposite linear attachment of muscles to the lips of bony crests and opposite areal attachment of muscles to either side of thin bony plates) suggests that in rare cases where precise anatomical architecture is such that net tension may be present, then bone is not found; such regions consist of appropriate collagenous structures. In the third situation (opposite attachment of tendons to a sesamoid bone) where it would appear that net tension ought to exist, it is suggested that it is likely that it does not. Net tension probably occurs only in tendons running in tightly curved bony grooves: those very situations where sesamoid bones are not present.