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.     

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.     

Thick filament movement and isometric tension in activated skeletal muscle.

Thick filaments can move from the center of the sarcomere to the Z-disc while the isometric tension remains stable in skinned rabbit psoas fibers activated for several minutes (Horowits and Podolsky, 1987). Using the active and resting tension-length relations and the force-velocity relation, we calculated the time course and mechanical consequences of thick filament movement in the presence and absence of the elastic titin filaments, which link the ends of the thick filaments to the Z-discs and give rise to the resting tension. The calculated time course of thick filament movement exhibits a lag phase, during which the velocity and extent of movement are extremely small. This lag phase is dependent only on the properties of the cross-bridges and the initial position of the thick filament. The time course of thick filament movement in skinned rabbit psoas fibers at 7 degrees C is well fit assuming a small initial thick filament displacement away from the center of the sarcomere; this leads to a lag of approximately 80 s before any significant thick filament movement occurs. In the model incorporating titin filaments, this lag is followed by a phase of slow, steady motion during which isometric tension is stable. The model excluding titin filaments predicts a phase of acceleration accompanied by a 50% decrease in tension. The observed time course of movement and tension are consistent with the model incorporating titin filaments. The long lag phase suggests that in vivo, significant movement of thick filaments is unlikely to occur during a single contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Passive tension in cardiac muscle: contribution of collagen, titin, microtubules, and intermediate filaments.

The passive tension-sarcomere length relation of rat cardiac muscle was investigated by studying passive (or not activated) single myocytes and trabeculae. The contribution of collagen, titin, microtubules, and intermediate filaments to tension and stiffness was investigated by measuring (1) the effects of KCl/KI extraction on both trabeculae and single myocytes, (2) the effect of trypsin digestion on single myocytes, and (3) the effect of colchicine on single myocytes. It was found that over the working range of sarcomeres in the heart (lengths approximately 1.9-2.2 microns), collagen and titin are the most important contributors to passive tension with titin dominating at the shorter end of the working range and collagen at longer lengths. Microtubules made a modest contribution to passive tension in some cells, but on average their contribution was not significant. Finally, intermediate filaments contributed about 10% to passive tension of trabeculae at sarcomere lengths from approximately 1.9 to 2.1 microns, and their contribution dropped to only a few percent at longer lengths. At physiological sarcomere lengths of the heart, cardiac titin developed much higher tensions (> 20-fold) than did skeletal muscle titin at comparable lengths. This might be related to the finding that cardiac titin has a molecular mass of 2.5 MDa, 0.3-0.5 MDa smaller than titin of mammalian skeletal muscle, which is predicted to result in a much shorter extensible titin segment in the I-band of cardiac muscle. Passive stress plotted versus the strain of the extensible titin segment showed that the stress-strain relationships are similar in cardiac and skeletal muscle. The difference in passive stress between cardiac and skeletal muscle at the sarcomere level predominantly resulted from much higher strains of the I-segment of cardiac titin at a given sarcomere length. By expressing a smaller titin isoform, without changing the properties of the molecule itself, cardiac muscle is able to develop significant levels of passive tension at physiological sarcomere lengths.     

Positive feedback by a potassium-selective inward rectifier enhances tuning in vertebrate hair cells.

Titin (also known as connectin) is a muscle-specific giant protein found inside the sarcomere, spanning from the Z-line to the M-line. The I-band segment of titin is considered to function as a molecular spring that develops tension when sarcomeres are stretched (passive tension). Recent studies on skeletal muscle indicate that it is not the entire I-band segment of titin that behaves as a spring; some sections are inelastic and do not take part in the development of passive tension. To better understand the mechanism of passive tension development in the heart, where passive tension plays an essential role in the pumping function, we investigated titin's elastic segment in cardiac myocytes using structural and mechanical techniques. Single cardiac myocytes were stretched by various amounts and then immunolabeled and processed for electron microscopy in the stretched state. Monoclonal antibodies that recognize different titin epitopes were used, and the locations of the titin epitopes in the sarcomere were studied as a function of sarcomere length. We found that only a small region of the I-band segment of titin is elastic; its contour length is estimated at approximately 75 nm, which is only approximately 40% of the total I-band segment of titin. Passive tension measurements indicated that the fundamental determinant of how much passive tension the heart develops is the strain of titin's elastic segment. Furthermore, we found evidence that in sarcomeres that are slack (length, approximately 1.85 microns) the elastic titin segment is highly folded on top of itself. Based on the data, we propose a two-stage mechanism of passive tension development in the heart, in which, between sarcomere lengths of approximately 1.85 microns and approximately 2.0 microns, titin's elastic segment straightens and, at lengths longer than approximately 2.0 microns, the molecular domains that make up titin's elastic segment unravel. Sarcomere shortening to lengths below slack (approximately 1.85 microns) also results in straightening of the elastic titin segment, giving rise to a force that opposes shortening and that tends to bring sarcomeres back to their slack length.     

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.     

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.     

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.     

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.     

Evidence for the oligomeric state of 'elastic' titin in muscle sarcomeres

The giant protein titin has important roles in muscle sarcomere integrity, elasticity and contractile activity. The key role in elasticity was highlighted in recent years by single-molecule mechanical studies, which showed a direct relationship between the non-uniform structure of titin and the hierarchical mechanism of its force-extension behavior. Further advances in understanding mechanisms controlling sarcomere structure and elasticity require detailed knowledge of titin arrangement and interactions in situ. Here we present data on the structure and self-interactive properties of an ∼ 290 kDa (∼ 100 nm long) tryptic fragment from the I-band part of titin that is extensible in situ. The fragment includes the conserved ‘distal’ tandem Ig segment of the molecule and forms side-by-side oligomers with distinctive 4 nm cross-striations. Comparisons between these oligomers and the end filaments seen at the tips of native thick filaments indicate identical structure. This shows that end-filaments are formed by the elastic parts of six titin molecules connecting each end of the thick filament to the Z-line. Self-association of elastic titin into stiff end-filaments adds a further hierarchical level in the mechanism of titin extensibility in muscle cells. Self-association of this part of titin may be required to prevent interference of the individual flexible molecules with myosin cross-bridges interacting with actin.     

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     

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.     

Proposed role of the M-band in sarcomere mechanics and mechano-sensing: a model study

In sarcomeres of striated muscles the middle parts of adjacent thick filaments are connected to each other by the M-band proteins. To understand the role of the M-band in sarcomere mechanics a model of forces which pull a thick filament to opposite Z-disks of a sarcomere is considered. Forces of actin-myosin cross-bridges, I-band titin segments and the M-band are accounted for. A continual expression for the M-band force is obtained assuming that the M-band proteins which connect neighbor thick filaments have nonlinear elastic properties. On the ascending and descending limbs of the force-length diagram cross-bridge forces tend to destabilize sarcomere while titin tries to restore its symmetric configuration. When destabilizing cross-bridge force exceeds a critical limit, symmetric configuration of a sarcomere becomes unstable and the M-band buckles. Stiffness of the M-band increases stability only if the M-band is anchored to the extra-sarcomere cytoskeleton. Realistic magnitudes of the M-band buckling require that the M-band proteins have essentially nonlinear elasticity. The buckling may explain the M-band bending and axial misalignment of the thick filaments observed in contracting muscle. We hypothesize that the buckling stretches the titin protein kinase domain localized in the M-band being the signal for mechanical control of gene expression and protein turnover in striated muscle.     

Titin elasticity and mechanism of passive force development in rat cardiac myocytes probed by thin-filament extraction.

Titin (also known as connectin) is a giant filamentous protein whose elastic properties greatly contribute to the passive force in muscle. In the sarcomere, the elastic I-band segment of titin may interact with the thin filaments, possibly affecting the molecule's elastic behavior. Indeed, several studies have indicated that interactions between titin and actin occur in vitro and may occur in the sarcomere as well. To explore the properties of titin alone, one must first eliminate the modulating effect of the thin filaments by selectively removing them. In the present work, thin filaments were selectively removed from the cardiac myocyte by using a gelsolin fragment. Partial extraction left behind approximately 100-nm-long thin filaments protruding from the Z-line, whereas the rest of the I-band became devoid of thin filaments, exposing titin. By applying a much more extensive gelsolin treatment, we also removed the remaining short thin filaments near the Z-line. After extraction, the extensibility of titin was studied by using immunoelectron microscopy, and the passive force-sarcomere length relation was determined by using mechanical techniques. Titin's regional extensibility was not detectably affected by partial thin-filament extraction. Passive force, on the other hand, was reduced at sarcomere lengths longer than approximately 2.1 microm, with a 33 +/- 9% reduction at 2.6 microm. After a complete extraction, the slack sarcomere length was reduced to approximately 1.7 microm. The segment of titin near the Z-line, which is otherwise inextensible, collapsed toward the Z-line in sarcomeres shorter than approximately 2.0 microm, but it was extended in sarcomeres longer than approximately 2.3 microm. Passive force became elevated at sarcomere lengths between approximately 1.7 and approximately 2.1 microm, but was reduced at sarcomere lengths of >2.3 microm. These changes can be accounted for by modeling titin as two wormlike chains in series, one of which increases its contour length by recruitment of the titin segment near the Z-line into the elastic pool.     

Lattice shrinkage with increasing resting tension in stretched, single skinned fibers of frog muscle.

The 1,0 lattice spacing d1,0 in chemically and mechanically skinned single fibers of frog muscle was measured at various sarcomere lengths, L, in the range from L = 2.1 to 6.0 microns by an x-ray diffraction method. In chemically skinned fibers, d1,0 decreased with a similar slope to that of mechanically skinned fibers up to L congruent to 3 microns, but beyond this point d1,0 steeply decreased with further stretching. This steep decrease in d1,0 could be ascribed mainly to an increase in the compressing force associated with the longitudinal extension of a remnant of the sarcolemma. In mechanically skinned fibers, the gradual decrease in d1,0 continued beyond filament overlap (L greater than or equal to 3.5 microns) and was highly proportional to a resting tension. This decrease in d1,0 at L greater than or equal to 3.5 microns could be ascribed to an increase in the force exerted by lateral elastic components, which is proportional to the longitudinal resting tension. A conceptual model is proposed of a network structure of elastic components in a sarcomere.     

Molecular mechanics of cardiac titin's PEVK and N2B spring elements.

Titin is a giant elastic protein that is responsible for the majority of passive force generated by the myocardium. Titin's force is derived from its extensible I-band region, which, in the cardiac isoform, comprises three main extensible elements: tandem Ig segments, the PEVK domain, and the N2B unique sequence (N2B-Us). Using atomic force microscopy, we characterized the single molecule force-extension curves of the PEVK and N2B-Us spring elements, which together are responsible for physiological levels of passive force in moderately to highly stretched myocardium. Stretch-release force-extension curves of both the PEVK domain and N2B-Us displayed little hysteresis: the stretch and release data nearly overlapped. The force-extension curves closely followed worm-like chain behavior. Histograms of persistence length (measure of chain bending rigidity) indicated that the single molecule persistence lengths are approximately 1.4 and approximately 0.65 nm for the PEVK domain and N2B-Us, respectively. Using these mechanical characteristics and those determined earlier for the tandem Ig segment (assuming folded Ig domains), we modeled the cardiac titin extensible region in the sarcomere and calculated the extension of the various spring elements and the forces generated by titin, both as a function of sarcomere length. In the physiological sarcomere length range, predicted values and those obtained experimentally were indistinguishable.     

Disassembly kinetics of thick filaments in rabbit skeletal muscle fibers. Effects of ionic strength, Ca2+ concentration, pH, temperature, and cross-bridges on the stability of thick filament structure.

The kinetics of dissociation from both ends of thick filaments in a muscle fiber was investigated by an optical diffraction method. The dissociation velocity of thick filaments at a sarcomere length of 2.75 microns increased with increasing the KCl concentration (from 60 mM to 0.5 M), increasing the pH value (from 6.2 to 8.0) or decreasing the temperature (from 25 to 5 degrees C) in the presence of 10 mM pyrophosphate and 5 mM MgCl2. Micromolar concentrations of Ca2+ suppressed the dissociation velocity markedly at shorter sarcomere lengths. The dissociation velocity, v, decreased as thick filaments became shorter, and v = -db/dt = vo exp (alpha b), where b is the length of the thick filament at time t and vo and alpha are constants. The vo value was largely dependent on the KCl concentration but the alpha value was not. The stiffness of a muscle fiber decreased nearly in proportion to the decrease of overlap between thick and thin filaments induced by the dissociation of thick filaments. This indicates that cross-bridges are uniformly distributed and contribute independently to the stiffness of a muscle fiber during the dissociation of thick filaments.     

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.     

Characterization of beta-connectin (titin 2) from striated muscle by dynamic light scattering.

Connectin (titin) is a large filamentous protein (single peptide) with a molecular mass of approximately 3 MDa, contour length approximately 900 nm, and diameter approximately 4 nm, and resides in striated muscle. Connectin links the thick filaments to the Z-lines in a sarcomere and produces a passive elastic force when muscle fiber is stretched. The aim of this study is to elucidate some aspects of physical properties of isolated beta-connectin (titin 2), a proteolytic fragment of connectin, by means of dynamic light-scattering (DLS) spectroscopy. The analysis of DLS spectra for beta-connectin gave the translational diffusion coefficient of 3.60 x 10(-8) cm2/s at 10 degrees C (or the hydrodynamic radius of 44.1 nm), molecular mass little smaller than 3.0 MDa (for a literature value of sedimentation coefficient), the root-mean-square end-to-end distance of 163 nm (or the radius of gyration of 66.6 nm), and the Kuhn segment number of 30 and segment length of 30 nm (or the persistence length of 15 nm). These results permitted to estimate the flexural rigidity of 6.0 x 10(-20) dyn x cm2 for filament bending, and the elastic constant of 7 dyn/cm for extension of one persistence length. Based on a simple model, implications of the present results in muscle physiology are discussed.     

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.