Passive force generation and titin isoforms in mammalian skeletal muscle.

When relaxed striated muscle cells are stretched, a resting tension is produced which is thought to arise from stretching long, elastic filaments composed of titin (also called connectin). Here, I show that single skinned rabbit soleus muscle fibers produce resting tension that is several-fold lower than that found in rabbit psoas fibers. At sarcomere lengths where the slope of the resting tension-sarcomere length relation is low, electron microscopy of skinned fibers indicates that thick filaments move from the center to the side of the sarcomere during prolonged activation. As sarcomeres are stretched and the resting tension sarcomere length relation becomes steeper, this movement is decreased. The sarcomere length range over which thick filament movement decreases is higher in soleus than in psoas fibers, paralleling the different lengths at which the slope of the resting tension-sarcomere length relations increase. These results indicate that the large differences in resting tension between single psoas and soleus fibers are due to different tensions exerted by the elastic elements linking the end of each thick filament to the nearest Z-disc, i.e., the titin filaments. Quantitative gel electrophoresis of proteins from single muscle fibers excludes the possibility that resting tension is less in soleus than in psoas fibers simply because they have fewer titin filaments. A small difference in the electrophoretic mobility of titin between psoas and soleus fibers suggests the alternate possibility that mammalian muscle cells use at least two titin isoforms with differing elastic properties to produce variations in resting tension.     

The positional stability of thick filaments in activated skeletal muscle depends on sarcomere length: evidence for the role of titin filaments

Electron microscopy was used to study the positional stability of thick filaments in isometrically contracting skinned rabbit psoas muscle as a function of sarcomere length at 7 degrees C. After calcium activation at a sarcomere length of 2.6 micron, where resting stiffness is low, sarcomeres become nonuniform in length. The dispersion in sarcomere length is complete by the time maximum tension is reached. A-bands generally move from their central position and continue moving toward one of the Z-discs after tension has reached a plateau at its maximum level. The lengths of the thick and thin filaments remain constant during this movement. The extent of A-band movement during contraction depends on the final length of the individual sarcomere. After prolonged activation, all sarcomeres between 1.9 and 2.5 micron long exhibit A-bands that are adjacent to a Z-disc, with no intervening I-band. Sarcomeres 2.6 or 2.7 micron long exhibit a partial movement of A-bands. At longer sarcomere lengths, where the resting stiffness exceeds the slope of the active tension-length relation, the A-bands remain perfectly centered during contraction. Sarcomere symmetry and length uniformity are restored upon relaxation. These results indicate that the central position of the thick filaments in the resting sarcomere becomes unstable upon activation. In addition, they provide evidence that the elastic titin filaments, which join thick filaments to Z-discs, produce almost all of the resting tension in skinned rabbit psoas fibers and act to resist the movement of thick filaments away from the center of the sarcomere during contraction.     

Viscoelasticity of the sarcomere matrix of skeletal muscles. The titin-myosin composite filament is a dual-stage molecular spring.

The mechanical roles of sarcomere-associated cytoskeletal lattices were investigated by studying the resting tension-sarcomere length curves of mechanically skinned rabbit psoas muscle fibers over a wide range of sarcomere strain. Correlative immunoelectron microscopy of the elastic titin filaments of the endosarcomeric lattice revealed biphasic extensibility behaviors and provided a structural interpretation of the multiphasic tension-length curves. We propose that the reversible change of contour length of the extensible segment of titin between the Z line and the end of thick filaments underlies the exponential rise of resting tension. At and beyond an elastic limit near 3.8 microns, a portion of the anchored titin segment that adheres to thick filaments is released from the distal ends of thick filament. This increase in extensible length of titin results in a net length increase in the unstrained extensible segment, thereby lowering the stiffness of the fiber, lengthening the slack sarcomere length, and shifting the yield point in postyield sarcomeres. Thus, the titin-myosin composite filament behaves as a dual-stage molecular spring, consisting of an elastic connector segment for normal response and a longer latent segment that is recruited at and beyond the elastic limit of the sarcomere. Exosarcomeric intermediate filaments contribute to resting tension only above 4.5 microns. We conclude that the interlinked endo- and exosarcomeric lattices are both viscoelastic force-bearing elements. These distinct cytoskeletal lattices appear to operate over two ranges of sarcomere strains and collectively enable myofibrils to respond viscoelastically over a broad range of sarcomere and fiber lengths.     

Passive tension and stiffness of vertebrate skeletal and insect flight muscles: the contribution of weak cross-bridges and elastic filaments.

Tension and dynamic stiffness of passive rabbit psoas, rabbit semitendinosus, and waterbug indirect flight muscles were investigated to study the contribution of weak-binding cross-bridges and elastic filaments (titin and minititin) to the passive mechanical behavior of these muscles. Experimentally, a functional dissection of the relative contribution of actomyosin cross-bridges and titin and minititin was achieved by 1) comparing mechanically skinned muscle fibers before and after selective removal of actin filaments with a noncalcium-requiring gelsolin fragment (FX-45), and 2) studying passive tension and stiffness as a function of sarcomere length, ionic strength, temperature, and the inhibitory effect of a carboxyl-terminal fragment of smooth muscle caldesmon. Our data show that weak bridges exist in both rabbit skeletal muscle and insect flight muscle at physiological ionic strength and room temperature. In rabbit psoas fibers, weak bridge stiffness appears to vary with both thin-thick filament overlap and with the magnitude of passive tension. Plots of passive tension versus passive stiffness are multiphasic and strikingly similar for these three muscles of distinct sarcomere proportions and elastic proteins. The tension-stiffness plot appears to be a powerful tool in discerning changes in the mechanical behavior of the elastic filaments. The stress-strain and stiffness-strain curves of all three muscles can be merged into one, by normalizing strain rate and strain amplitude of the extensible segment of titin and minititin, further supporting the segmental extension model of resting tension development.     

Elastic behavior of connectin filaments during thick filament movement in activated skeletal muscle

Connectin (also called titin) is a huge, striated muscle protein that binds to thick filaments and links them to the Z-disc. Using an mAb that binds to connectin in the I-band region of the molecule, we studied the behavior of connectin in both relaxed and activated skinned rabbit psoas fibers by immunoelectron microscopy. In relaxed fibers, antibody binding is visualized as two extra striations per sarcomere arranged symmetrically about the M-line. These striations move away from both the nearest Z-disc and the thick filaments when the sarcomere is stretched, confirming the elastic behavior of connectin within the I- band of relaxed sarcomeres as previously observed by several investigators. When the fiber is activated, thick filaments in sarcomeres shorter than 2.8 microns tend to move from the center to the side of the sarcomere. This translocation of thick filaments within the sarcomere is accompanied by movement of the antibody label in the same direction. In that half-sarcomere in which the thick filaments move away from the Z-disc, the spacings between the Z-disc and the antibody and between the antibody and the thick filaments both increase. Conversely, on the side of the sarcomere in which the thick filaments move nearer to the Z-line, these spacings decrease. Regardless of whether I-band spacing is varied by stretch of a relaxed sarcomere or by active sliding of thick filaments within a sarcomere of constant length, the spacings between the Z-line and the antibody and between the antibody and the thick filaments increase with I-band length identically. These results indicate that the connectin filaments remain bound to the thick filaments in active fibers, and that the elastic properties of connectin are unaltered by calcium ions and cross-bridge activity.     

Compliance of thin filaments in skinned fibers of rabbit skeletal muscle.

The mechanical compliance (reciprocal of stiffness) of thin filaments was estimated from the relative compliance of single, skinned muscle fibers in rigor at sarcomere lengths between 1.8 and 2.4 micron. The compliance of the fibers was calculated as the ratio of sarcomere length change to tension change during imposition of repetitive cycles of small stretches and releases. Fiber compliance decreased as the sarcomere length was decreased below 2.4 micron. The compliance of the thin filaments could be estimated from this decrement because in this range of lengths overlap between the thick and thin filaments is complete and all of the myosin heads bind to the thin filament in rigor. Thus, the compliance of the overlap region of the sarcomere is constant as length is changed and the decrease in fiber compliance is due to decrease of the nonoverlap length of the thin filaments (the I band). The compliance value obtained for the thin filaments implies that at 2.4-microns sarcomere length, the thin filaments contribute approximately 55% of the total sarcomere compliance. Considering that the sarcomeres are approximately 1.25-fold more compliant in active isometric contractions than in rigor, the thin filaments contribute approximately 44% to sarcomere compliance during isometric contraction.     

Stiffness of glycerinated rabbit psoas fibers in the rigor state. Filament-overlap relation

The stiffness of glycerinated rabbit psoas fibers in the rigor state was measured at various sarcomere lengths in order to determine the distribution of the sarcomere compliance between the cross-bridge and other structures. The stiffness was determined by measuring the tension increment at one end of a fiber segment while stretching the other end of the fiber. The contribution of the end compliance to the rigor segments was checked both by laser diffractometry of the sarcomere length change and by measuring the length dependence of the Young's modulus; the contribution was found to be small. The stiffness in the rigor state was constant at sarcomere lengths of 2.4 microns or less; at greater sarcomere lengths the stiffness, when corrected for the contribution of resting stiffness, scaled with the amount of overlap between the thick and thin filaments. These results suggest that the source of the sarcomere compliance of the rigor fiber at the full overlapping of filaments is mostly the cross-bridge compliance.     

Elastic filaments in skeletal muscle revealed by selective removal of thin filaments with plasma gelsolin

Muscle needs an elastic framework to maintain its mechanical stability. Removal of thin filaments in rabbit skeletal muscle with plasma gelsolin has revealed the essential features of elastic filaments. The selective removal of thin filaments was confirmed by staining with phalloidin-rhodamine for fluorescence microscopy, examination of arrowhead formation with myosin subfragment 1 by electron microscopy, and analysis by SDS-PAGE. Thin section electron microscopy revealed the elastic fine filaments (approximately 4 nm in diameter) connecting thick filaments and the Z line. After removal of thin filaments, both rigor stiffness and active tension generation were lost, but the resting tension remained. These observations indicate that the thin filament-free fibers maintain a framework composed of the serial connections of thick filaments, the elastic filaments, and the Z line, which gives passive elasticity to the contractile system of skeletal muscle. The resting tension that remained in the thin filament-free fibers was decreased by mild trypsin treatment. The only protein component that was digested in parallel with the decrease in the resting tension and the disappearance of the elastic filaments was alpha-connectin (also called titin 1), which was transformed from the alpha to the beta form (from titin 1 to 2, respectively). Thus, we conclude that the main protein component of the elastic filaments is alpha-connectin (titin 1).     

Alterations in Ca2+ sensitive tension due to partial extraction of C- protein from rat skinned cardiac myocytes and rabbit skeletal muscle fibers

C-protein, a substantial component of muscle thick filaments, has been postulated to have various functions, based mainly on results from biochemical studies. In the present study, effects on Ca(2+)-activated tension due to partial removal of C-protein were investigated in skinned single myocytes from rat ventricle and rabbit psoas muscle. Isometric tension was measured at pCa values of 7.0 to 4.5: (a) in untreated myocytes, (b) in the same myocytes after partial extraction of C-protein, and (c) in some myocytes, after readdition of C-protein. The solution for extracting C-protein contained 10 mM EDTA, 31 mM Na2HPO2, 124 mM NaH2PO4, pH 5.9 (Offer et al., 1973; Hartzell and Glass, 1984). In addition, the extracting solution contained 0.2 mg/ml troponin and, for skeletal muscle, 0.2 mg/ml myosin light chain-2 in order to minimize loss of these proteins during the extraction procedure. Between 60 and 70% of endogenous C-protein was extracted from cardiac myocytes by a 1-h soak in extracting solution at 20-23 degrees C; a similar amount was extracted from psoas fibers during a 3-h soak at 25 degrees C. For both cardiac myocytes and skeletal muscle fibers, partial extraction of C-protein resulted in increased active tension at submaximal concentrations of Ca2+, but had little effect upon maximum tension. C-protein extraction also reduced the slope of the tension-pCa relationships, suggesting that the cooperativity of Ca2+ activation of tension was decreased. Readdition of C-protein to previously extracted myocytes resulted in recovery of both tension and slope to near their control values. The effects on tension did not appear to be due to disruption of cooperative activation of the thin filament, since C-protein extraction from cardiac myocytes that were 40-60% troponin-C (TnC) deficient produced effects similar to those observed in cells that were TnC replete. Measurements of the tension-pCa relationship in skeletal muscle fibers were also made at a sarcomere length of 3.5 microns which, because of the distribution of C-protein on the thick filament, should eliminate any interaction between C-protein and actin. The effects of C-protein extraction were similar at long and short sarcomere lengths. These data are consistent with a model in which C-protein modulates the range of movement of myosin, such that the probability of myosin binding to actin is increased after its extraction.     

A simple model with myofilament compliance predicts activation-dependent crossbridge kinetics in skinned skeletal fibers

The contribution of thick and thin filaments to skeletal muscle fiber compliance has been shown to be significant. If similar to the compliance of cycling cross-bridges, myofilament compliance could explain the difference in time course of stiffness and force during the rise of tension in a tetanus as well as the difference in Ca(2+) sensitivity of force and stiffness and more rapid phase 2 tension recovery (r) at low Ca(2+) activation. To characterize the contribution of myofilament compliance to sarcomere compliance and isometric force kinetics, the Ca(2+)-activation dependence of sarcomere compliance in single glycerinated rabbit psoas fibers, in the presence of ATP (5.0 mM), was measured using rapid length steps. At steady sarcomere length, the dependence of sarcomere compliance on the level of Ca(2+)-activated force was similar in form to that observed for fibers in rigor where force was varied by changing length. Additionally, the ratio of stiffness/force was elevated at lower force (low [Ca(2+)]) and r was faster, compared with maximum activation. A simple series mechanical model of myofilament and cross-bridge compliance in which only strong cross-bridge binding was activation dependent was used to describe the data. The model fit the data and predicted that the observed activation dependence of r can be explained if myofilament compliance contributes 60-70% of the total fiber compliance, with no requirement that actomyosin kinetics be [Ca(2+)] dependent or that cooperative interactions contribute to strong cross-bridge binding.     

Titin and the sarcomere symmetry paradox

Titin is thought to play a major role in myofibril assembly, elasticity and stability. A single molecule spans half the sarcomere and makes interactions with both a thick filament and the Z-line. In the unit cell structure of each half sarcomere there is one thick filament with 3-fold symmetry and two thin filaments with approximately 2-fold symmetry. The minimum number of titin molecules that could satisfy both these symmetries is 12. We determined the actual number of titin molecules in a unit cell from scanning transmission electron microscopy mass measurements of end-filaments. One of these emerges from each tip of the thick filament and is thought to be the in-register aggregate of the titin molecules associated with the filament. The mass per unit length of the end-filament (17.1 kDa/nm) is consistent with six titin molecules not 12. Thus the number of titin molecules present is insufficient to satisfy both symmetries. We suggest a novel solution to this paradox in which four of the six titin molecules interact with the two thin filaments in the unit cell, while the remaining two interact with the two thin filaments that enter the unit cell from the adjacent sarcomere. This arrangement would augment mechanical stability in the sarcomere.     

Architecture of the sarcomere matrix of skeletal muscle: immunoelectron microscopic evidence that suggests a set of parallel inextensible nebulin filaments anchored at the Z line

Nebulin, a giant myofibrillar protein (600-800 kD) that is abundant (3%) in the sarcomere of a wide range of skeletal muscles, has been proposed as a component of a cytoskeletal matrix that coexists with actin and myosin filaments within the sarcomere. Immunoblot analysis indicates that although polypeptides of similar size are present in cardiac and smooth muscles at low abundance, those proteins show no immunological cross-reactivity with skeletal muscle nebulin. Gel analysis reveals that nebulins in various skeletal muscles of rabbit belong to at least two classes of size variants. A monospecific antibody has been used to localize nebulin by immunoelectron microscopy in a mechanically split rabbit psoas muscle fiber preparation. Labeled split fibers exhibit six pairs of stripes of antibody-imparted transverse densities spaced at 0.1-1.0 micron from the Z line within each sarcomere. These epitopes maintain a fixed distance to the Z line irrespective of sarcomere length and do not exhibit the characteristic elastic stretch-response of titin epitopes within the I band domain. It is proposed that nebulin constitutes a set of inextensible filaments attached at one end to the Z line and that nebulin filaments are in parallel, and not in series, with titin filaments. Thus the skeletal muscle sarcomere may have two sets of nonactomyosin filaments: a set of I segment-linked nebulin filaments and a set of A segment-linked titin filaments. This four-filament sarcomere model raises the possibility that nebulin and titin might act as organizing templates and length- determining factors for actin and myosin respectively.     

Expression of titin isoforms in red and white muscle fibres of carp (Cyprinus carpio L.) exposed to different sarcomere strains during swimming

Titin (also known as connectin) is a striated-muscle-specific protein that spans the distance between the Z- and M-lines of the sarcomere. The elastic segment of the titin molecule in the I-band is thought to be responsible for developing passive tension and for maintaining the central position of thick filaments in contracting sarcomeres. Different muscle types express isoforms of titin that differ in their molecular mass. To help to elucidate the relation between the occurrence of titin isoforms and the functional properties of different fibre types, we investigated the presence of different titin isoforms in red and white fibres of the axial muscles of carp. Gel electrophoresis of single fibres revealed that the molecular mass of titin was larger in red than in white fibres. Fibres from anterior and posterior axial muscles were also compared. For both white and red fibres the molecular mass of titin in posterior muscle fibres was larger than in anterior muscle fibres. Thus, the same fibre type can express different titin isoforms depending on its location along the body axis. The contribution of titin to passive tension and stiffness of red anterior and posterior fibres was also determined. Single fibres were skinned and the sarcomere length dependencies of passive tension and passive stiffness were determined. Measurements were made before and after extracting thin and thick filaments using relaxing solutions with 0.6 mol · l⁻¹ KCl and 1 mol · l⁻¹ KI. Tension and stiffness measured before extraction were assumed to result from both titin and intermediate filaments, and tension after extraction from only intermediate filaments. Compared to mammalian skeletal muscle, intermediate filaments developed high levels of tension and stiffness in both posterior and anterior fibres. The passive tension-sarcomere length curve of titin increased more steeply in red anterior fibres than in red posterior fibres and the curve reached a plateau at a shorter sarcomere length. Thus, the smaller titin isoform of anterior fibres results in more passive tension and stiffness for a given sarcomere strain. During continuous swimming, red fibres are exposed to larger changes in sarcomere strain than white fibres, and posterior fibres to larger changes in strain than anterior fibres. We propose that sarcomere strain is one of the functional parameters that modulates the expression of different titin isoforms in axial muscle fibres of carp. Accepted: 7 May 1997     

Calcium-dependent inhibition of in vitro thin-filament motility by native titin

Titin (also known as connectin) is a giant filamentous protein that spans the distance between the Z- and M-lines of the vertebrate muscle sarcomere and plays a fundamental role in the generation of passive tension. Titin has been shown to bind strongly to myosin, making it tightly associated to the thick filament in the sarcomere. Recent observations have suggested the possibility that titin also interacts with actin, implying further functions of titin in muscle contraction. We show — using in vitro motility and binding assays — that native titin interacts with both filamentous actin and reconstituted thin filaments. The interaction results in the inhibition of the filaments' in vitro motility. Furthermore, the titin-thin filament interaction occurs in a calcium-dependent manner: increased calcium results in enhanced binding of thin filaments to titin and greater suppression of in vitro motility.     

ULTRASTRUCTURE OF THE RESTING AND CONTRACTED STRIATED MUSCLE FIBER AT DIFFERENT DEGREES OF STRETCH

Passive stretch, isometric contraction, and shortening were studied in electron micrographs of striated, non-glycerinated frog muscle fibers. The artifacts due to the different steps of preparation were evaluated by comparing sarcomere length and fiber diameter before, during, and after fixation and after sectioning. Tension and length were recorded in the resting and contracted fiber before and during fixation. The I filaments could be traced to enter the A band between the A filaments on both sides of the I band, creating a zone of overlap which decreased linearly with stretch and increased with shortening. This is consistent with a sliding filament model. The decrease in the length of the A and I filaments during isometric contraction and the finding that fibers stretched to a sarcomere length of 3.7 µ still developed 30 per cent of the maximum tetanic tension could not be explained in terms of the sliding filament model. Shortening of the sarcomeres near the myotendinous junctions which still have overlap could account for only one-sixth of this tension, indicating that even those sarcomeres stretched to such a degree that there is a gap between A and I filaments are activated during isometric contraction (increase in stiffness). Shortening, too, was associated with changes in filament length. The diameter of A filaments remained unaltered with stretch and with isometric contraction. Shortening of 50 per cent was associated with a 13 per cent increase in A filament diameter. The area occupied by the fibrils and by the interfibrillar space increased with shortening, indicating a 20 per cent reduction in the volume of the fibrils when shortening amounted to 40 per cent.     

Thick-filament strain and interfilament spacing in passive muscle: effect of titin-based passive tension

We studied the effect of titin-based passive tension on sarcomere structure by simultaneously measuring passive tension and low-angle x-ray diffraction patterns on passive fiber bundles from rabbit skinned psoas muscle. We used a stretch-hold-release protocol with measurement of x-ray diffraction patterns at various passive tension levels during the hold phase before and after passive stress relaxation. Measurements were performed in relaxing solution without and with dextran T-500 to compress the lattice toward physiological levels. The myofilament lattice spacing was measured in the A-band (d_1,0) and Z-disk (d_Z) regions of the sarcomere. The axial spacing of the thick-filament backbone was determined from the sixth myosin meridional reflection (M6) and the equilibrium positions of myosin heads from the fourth myosin layer line peak position and the I_1,1/I_1,0 intensity ratio. Total passive tension was measured during the x-ray experiments, and a differential extraction technique was used to determine the relations between collagen- and titin-based passive tension and sarcomere length. Within the employed range of sarcomere lengths (∼2.2–3.4 μm), titin accounted for >80% of passive tension. X-ray results indicate that titin compresses both the A-band and Z-disk lattice spacing with viscoelastic behavior when fibers are swollen after skinning, and elastic behavior when the lattice is reduced with dextran. Titin also increases the axial thick-filament spacing, M6, in an elastic manner in both the presence and absence of dextran. No changes were detected in either I_1,1/I_1,0 or the position of peaks on the fourth myosin layer line during passive stress relaxation. Passive tension and M6 measurements were converted to thick-filament compliance, yielding a value of ∼85 m/N, which is several-fold larger than the thick-filament compliance determined by others during the tetanic tension plateau of activated intact muscle. This difference can be explained by the fact that thick filaments are more compliant at low tension (passive muscle) than at high tension (tetanic tension). The implications of our findings are discussed.     

Lattice swelling with the selective digestion of elastic components in single-skinned fibers of frog muscle.

Changes in the 1.0 lattice spacing during trypsin (0.25 micrograms/ml) treatment in mechanically skinned single fibers of frog muscle was examined by an x-ray diffraction method at various sarcomere lengths. The resting tension of a relaxed fiber was decreased by trypsin treatment but the stiffness of a rigor fiber was not, suggesting that elastic components were selectively digested. With progression of the digestion, the lattice spacing increased remarkably at longer sarcomere lengths and finally became independent of the sarcomere length. The increase in the lattice spacing was proportional to the decrease in the resting tension. These results support our previous suggestion (Higuchi, H., and Y. Umazume, 1986, Biophys. J., 50:385-389) that the lattice spacing decreases at long lengths due to compressive force exerted by a lateral elastic component that connects thick filaments to an axial elastic component. Consequently, it is unlikely that the decrease in the lattice spacing is determined by a decrease in the repulsive force between thick and thin filaments with stretching a fiber.     

The structure of thick filaments on longitudinal sections of rabbit psoas muscle

By means of electron microscopy the longitudinal sections of chemically skinned fibres of rigorised rabbit psoas muscle have been examined at pH of rigorising solutions equal to 6, 7, 8 (I = 0.125) and ionic strengths equal to 0.04, 0.125, 0.34 (pH 7.0). It has been revealed that at pH 6.0 the bands of minor proteins localization in A-disks were seen very distinctly, while at pH 7.0 and I = 0.125 these bands can be revealed only by means of antibody labelling technique. At the ionic strength of 0.34 (pH 7.0) the periodicity of 14.3 nm in thick filaments was clearly observed, which was determined by packing of the myosin rods into the filament shaft and of the myosin heads (cross-bridges) on the filament surface. The number of cross-bridge rows in the filament equals 102. A new scheme of myosin cross-bridge distribution in thick filaments of rabbit psoas muscle has been suggested according to which two rows of cross-bridges at each end of a thick filament are absent. The filament length equals 1.64 +/- 0.01 micron. It has been shown that the length of thick filament as well as the structural organization of their end regions in rabbit psoas muscle and frog sartorius one are different.     

Extensibility of the myofilaments in vertebrate skeletal muscle as revealed by stretching rigor muscle fibers

The extensibility of the myofilaments in vertebrate skeletal muscle was studied by stretching glycerinated rabbit psoas muscle fibers in rigor state and examining the resulting extension of sarcomere structures under an electron microscope. Although stretches applied to rigor fibers produced a successive yielding of the weakest sarcomeres, the length of the remaining intact sarcomeres in many myofibrils was fairly uniform, being definitely longer than the sarcomeres in the control, nonstretched part of rigor fibers. The stretch-induced increase in sarcomere length was found to be taken up by the extension of the H zone and the I band, whereas the amount of overlap between the thick and thin filaments did not change appreciably with stretches of 10-20%. The thick filament extension in the H zone was localized in the bare regions, whereas the thin filament extension in the I band appeared to take place uniformly along the filament length. No marked increase in the Z-line width was observed even with stretches of 20-30%. These results clearly demonstrate the extensibility of the thick and thin filaments. The possible contribution of the myofilament compliance to the series elastic component (SEC) in vertebrate skeletal muscle fibers is discussed on the basis of the electron microscopic data and the force-extension curve of the SEC in rigor fibers.     

Passive tension of rat skeletal soleus muscle fibers: effects of unloading conditions.

In this work we studied changes in passive elastic properties of rat soleus muscle fibers subjected to 14 days of hindlimb unloading (HU). For this purpose, we investigated the titin isoform expression in soleus muscles, passive tension-fiber strain relationships of single fibers, and the effects of the thick filament depolymerization on passive tension development. The myosin heavy chain composition was also measured for all fibers studied. Despite a slow-to-fast transformation of the soleus muscles on the basis of their myosin heavy chain content, no modification in the titin isoform expression was detected after 14 days of HU. However, the passive tension-fiber strain relationships revealed that passive tension of both slow and fast HU soleus fibers increased less steeply with sarcomere length than that of control fibers. Gel analysis suggested that this result could be explained by a decrease in the amount of titin in soleus muscle after HU. Furthermore, the thick filament depolymerization was found to similarly decrease passive tension in control and HU soleus fibers. Taken together, these results suggested that HU did not change titin isoform expression in the soleus muscle, but rather modified muscle stiffness by decreasing the amount of titin.