Force enhancement above the initial isometric force on the descending limb of the force-length relationship

Edman et al. (J. General Physiol. 80 (1982) 769) observed in single fibres of frog that the steady-state forces following active fibre stretch were greater than the purely isometric force obtained at the length from which the stretch was initiated. Operating on the descending limb of the force-length relationship, such a result can only be explained within the framework of the sarcomere length non-uniformity theory, if some fibre segments shortened during the fibre stretch. However, such a result was not found, leaving Edman's observation unexplained. Force enhancement above the initial isometric force has not been investigated systematically in whole muscle, and therefore it is not known whether this property is also part of whole muscle mechanics. The purpose of this study was to test if the steady-state forces following active stretch of cat semitendinosus were greater than the corresponding purely isometric forces at the muscle length from which the stretch was started. Cat semitendinosus was stretched by various amounts on the descending limb of the force-length relationship, and the steady-state forces following these stretches were compared to the corresponding isometric forces at the initial and final muscle lengths. In 109 of 131 tests, the steady-state forces following stretching were greater than the isometric forces at the initial muscle lengths. Force enhancement increased with increasing amounts of stretching, and force enhancement above the initial isometric force was more likely to occur following stretches of great compared to small amplitude. Passive forces following active muscle stretching were often significantly greater than the passive forces at the same muscle length following an isometric contraction or a passive stretching of the muscle. This observation was made consistently at the longest muscle lengths tested. It appears, therefore, that there is a passive force that accounts for part of the force enhancement above the isometric force at the initial muscle length, and that provides increased passive force when a muscle is actively, rather than passively, stretched at long muscle lengths. We conclude that cat semitendinosus demonstrates steady-state force enhancement above the corresponding purely isometric force at the initial muscle length on the descending limb of the force-length relationship for many contractile conditions, and that a unique, and so far undetected, passive, parallel element contributes to this force enhancement, particularly at long muscle lengths where muscle is assumed to be most vulnerable to injuries associated with sarcomere length instability.     

The effects of muscle stretching and shortening on isometric forces on the descending limb of the force-length relationship

The purpose of this study was to examine the effects of stretching and shortening on the isometric forces at different lengths on the descending limb of the force-length relationship. Cat soleus (N = 10) was stretched and shortened by various amounts on the descending limb of the force-length relationship, and the steady-state forces following these dynamic contractions were compared to the isometric forces at the corresponding muscle lengths. We found a shift of the force-length relationship to greater force values following muscle stretching, and to smaller force values following muscle shortening. Shifts in both directions critically depended on the magnitude of stretching/shortening and the final muscle length. We confirm recent findings that the steady-state isometric force following some stretch conditions clearly exceeded the maximal isometric forces at optimum muscle length, and that force enhancement was associated with an increase in the passive force, i.e., a passive force enhancement. When the passive force enhancement was subtracted from the total force enhancement, forces following stretch were always equal to or smaller than the isometric force at optimum muscle length. Together, these findings led to the conclusions: (a). that force enhancement is composed of an "active and a "passive" component; (b). that the "passive" component of force enhancement allows for forces greater than the maximal isometric forces at the muscle's optimum length; and (c). that force enhancement and force depression are critically affected by muscle length and stretch/shortening amplitude.     

Residual force enhancement exceeds the isometric force at optimal sarcomere length for optimized stretch conditions.

Residual force enhancement (FE) following stretch of an activated muscle is a well accepted property of skeletal muscle contraction. However, the mechanism underlying FE remains unknown. A crucial assumption on which some proposed mechanisms are based is the idea that forces in the enhanced state cannot exceed the steady-state isometric force at a sarcomere length associated with optimal myofilament overlap. Although there are a number of studies in which forces in the enhanced state were compared with the corresponding isometric forces on the plateau of the force-length relationship, these studies either did not show enhanced forces above the plateau or, if they did, they lacked measurements of sarcomere lengths confirming the plateau region. Here, we revisited this question by optimizing stretch conditions and measuring the average sarcomere lengths in isolated fibers, and we found that FE exceeded the maximal isometric reference force obtained at the plateau of the force-length relationship consistently (mean+/-SD: 4.8+/-2.1%) and by up to 10%. When subtracting the passive component of FE from the total FE, the enhanced forces remained greater than the isometric plateau force (mean+/-SD: 4.3+/-2.0%). Calcium-induced increases in passive forces, known to be present in single fibers and myofibrils, are too small to account for the FE observed here. We conclude that FE cannot be explained exclusively with a stretch-induced development of sarcomere length nonuniformities, that FE in single fibers may be associated with the recruitment of additional contractile force, and that isometric steady-state forces in the enhanced state are not uniquely determined by sarcomere lengths.     

Force enhancement following an active stretch in skeletal muscle

When skeletal muscle is stretched during a tetanic contraction, the resulting force is greater than the purely isometric force obtained at the corresponding final length. Several mechanisms have been proposed to explain this phenomenon, but the most accepted mechanism is the sarcomere length non-uniformity theory. This theory is associated with the notion of instability of sarcomeres on the descending limb of the force–length relationship. However, recent evidence suggests that this theory cannot account solely for the stretch-induced force enhancement. Some of this evidence is presented in this paper, and a new mechanism for force enhancement is proposed: one that is associated with the engagement of a passive force during stretch. We speculate that this passive force enhancement may be caused by titin, a protein associated with passive force production at long sarcomere lengths.     

Stretch-induced,steady-state force enhancement in single skeletal muscle fibers exceeds the isometric force at optimum fiber length

Stretch-induced force enhancement has been observed in a variety of muscle preparations and on structural levels ranging from single fibers to in vivo human muscles. It is a well-accepted property of skeletal muscle. However, the mechanism causing force enhancement has not been elucidated, although the sarcomere-length non-uniformity theory has received wide support. The purpose of this paper was to re-investigate stretch-induced force enhancement in frog single fibers by testing specific hypotheses arising from the sarcomere-length non-uniformity theory. Single fibers dissected from frog tibialis anterior (TA) and lumbricals (n=12 and 22, respectively) were mounted in an experimental chamber with physiological Ringer's solution (pH=7.5) between a force transducer and a servomotor length controller. The tetantic force-length relationship was determined. Isometric reference forces were determined at optimum length (corresponding to the maximal, active, isometric force), and at the initial and final lengths of the stretch experiments. Stretch experiments were performed on the descending limb of the force-length relationship after maximal tetanic force was reached. Stretches of 2.5-10% (TA) and 5-15% lumbricals of fiber length were performed at 0.1-1.5 fiber lengths/s. The stretch-induced, steady-state, active isometric force was always equal or greater than the purely isometric force at the muscle length from which the stretch was initiated. Moreover, for stretches of 5% fiber length or greater, and initiated near the optimum length of the fiber, the stretch-enhanced active force always exceeded the maximal active isometric force at optimum length. Finally, we observed a stretch-induced enhancement of passive force. We conclude from these results that the sarcomere length non-uniformity theory alone cannot explain the observed force enhancement, and that part of the force enhancement is associated with a passive force that is substantially greater after active compared to passive muscle stretch.     

A multi-scale continuum model of skeletal muscle mechanics predicting force enhancement based on actin–titin interaction

Although recent research emphasises the possible role of titin in skeletal muscle force enhancement, this property is commonly ignored in current computational models. This work presents the first biophysically based continuum-mechanical model of skeletal muscle that considers, in addition to actin–myosin interactions, force enhancement based on actin–titin interactions. During activation, titin attaches to actin filaments, which results in a significant reduction in titin’s free molecular spring length and therefore results in increased titin forces during a subsequent stretch. The mechanical behaviour of titin is included on the microscopic half-sarcomere level of a multi-scale chemo-electro-mechanical muscle model, which is based on the classic sliding-filament and cross-bridge theories. In addition to titin stress contributions in the muscle fibre direction, the continuum-mechanical constitutive relation accounts for geometrically motivated, titin-induced stresses acting in the muscle’s cross-fibre directions. Representative simulations of active stretches under maximal and submaximal activation levels predict realistic magnitudes of force enhancement in fibre direction. For example, stretching the model by 20 % from optimal length increased the isometric force at the target length by about 30 %. Predicted titin-induced stresses in the muscle’s cross-fibre directions are rather insignificant. Including the presented development in future continuum-mechanical models of muscle function in dynamic situations will lead to more accurate model predictions during and after lengthening contractions.     

Force enhancement and relaxation rates after stretch of activated muscle fibres

The residual force enhancement following muscle stretch might be associated with an increase in the proportion of attached cross-bridges, as supported by stiffness measurements. In this case, it could be caused by an increase in the attachment or a decrease in the detachment rate of cross-bridges, or a combination of the two. The purpose of this study was to investigate if the stretch-induced force enhancement is related to cross-bridge attachment/detachment kinetics. Single muscle fibres dissected from the lumbrical muscle of frog were place at a length approximately 20% longer than the plateau of the force-length relationship; they were maximally activated, and after full isometric force was reached, ramp stretches were imposed with amplitudes of 5 and 10% fibre length, at a speed of 40% fibre length s(-1). Experiments were performed in Ringer's solution, and with the addition of 2, 5 and 10 nM of 2,3-butanedione monoxime (BDM), a drug that places cross-bridges in a pre-power-stroke, state, inhibiting force production. The total force following stretch was higher than the corresponding force measured after isometric contraction at the corresponding length. This residual force enhancement was accompanied by an increase relaxation time. BDM, which decreases force production during isometric contractions, considerably increased the relative levels of force enhancement. BDM also increased relaxation times after stretch, beyond the levels observed during reference contractions in Ringer's solution, and beyond isometric control tests at the corresponding BDM concentrations. Together, these results support the idea that force enhancement is caused, at least in part, by a decrease in cross-bridge detachment rates, as manifested by the increased relaxation times following fibre stretch.     

Residual force enhancement in myofibrils and sarcomeres

Residual force enhancement has been observed following active stretch of skeletal muscles and single fibres. However, there has been intense debate whether force enhancement is a sarcomeric property, or is associated with sarcomere length instability and the associated development of non-uniformities. Here, we studied force enhancement for the first time in isolated myofibrils (n=18) that, owing to the strict in series arrangement, allowed for evaluation of this property in individual sarcomeres (n=79). We found consistent force enhancement following stretch in all myofibrils and each sarcomere, and forces in the enhanced state typically exceeded the isometric forces on the plateau of the force-length relationship. Measurements were made on the plateau and the descending limb of the force-length relationship and revealed gross sarcomere length non-uniformities prior to and following active myofibril stretching, but in contrast to previous accounts, revealed that sarcomere lengths were perfectly stable under these experimental conditions. We conclude that force enhancement is a sarcomeric property that does not depend on sarcomere length instability, that force enhancement varies greatly for different sarcomeres within the same myofibril and that sarcomeres with vastly different amounts of actin-myosin overlap produce the same isometric steady-state forces. This last finding was not explained by differences in the amount of contractile proteins within sarcomeres, vastly different passive properties of individual sarcomeres or (half-) sarcomere length instabilities, suggesting that the basic mechanical properties of muscles, such as force enhancement, force depression and creep, which have traditionally been associated with sarcomere instabilities and the corresponding dynamic redistribution of sarcomere lengths, are not caused by such instabilities, but rather seem to be inherent properties of the mechanisms of contraction.     

Active force inhibition and stretch-induced force enhancement in frog muscle treated with BDM.

There is evidence that the stretch-induced residual force enhancement observed in skeletal muscles is associated with 1) cross-bridge dynamics and 2) an increase in passive force. The purpose of this study was to characterize the total and passive force enhancement and to evaluate whether these phenomena may be associated with a slow detachment of cross bridges. Single fibers from frog lumbrical muscles were placed at a length 20% longer than the plateau of the force-length relationship, and active and passive stretches (amplitudes of 5 and 10% of fiber length and at a speed of 40% fiber length/s) were performed. Experiments were conducted in Ringer solution and with the addition of 2, 5, and 10 mM of 2,3-butanedione monoxime (BDM), a cross-bridge inhibitor. The steady-state active and passive isometric forces after stretch of an activated fiber were higher than the corresponding forces measured after isometric contractions or passive stretches. BDM decreased the absolute isometric force and increased the total force enhancement in all conditions investigated. These results suggest that total force enhancement is directly associated with cross-bridge kinetics. Addition of 2 mM BDM did not change the passive force enhancement after 5 and 10% stretches. Addition of 5 and 10 mM did not change (5% stretches) or increased (10% stretches) the passive force enhancement. Increasing stretch amplitudes and increasing concentrations of BDM caused relaxation after stretch to be slower, and because passive force enhancement is increased at the greatest stretch amplitudes and the highest BDM concentrations, it appears that passive force enhancement may be related to slow-detaching cross bridges.     

Are titin properties reflected in single myofibrils?

Titin is a structural protein in muscle that spans the half sarcomere from Z-band to M-line. Although there are selected studies on titin's mechanical properties from tests on isolated molecules or titin fragments, little is known about its behavior within the structural confines of a sarcomere. Here, we tested the hypothesis that titin properties might be reflected well in single myofibrils. Single myofibrils from rabbit psoas were prepared for measurement of passive stretch-shortening cycles at lengths where passive titin forces occur. Three repeat stretch-shortening cycles with magnitudes between 1.0 and 3.0μm/sarcomere were performed at a speed of 0.1μm/s·sarcomere and repeated after a ten minute rest at zero force. These tests were performed in a relaxation solution (passive) and an activation solution (active) where cross-bridge attachment was inhibited with 2,3 butanedionemonoxime. Myofibrils behaved viscoelastically producing an increased efficiency with repeat stretch-shortening cycles, but a decreased efficiency with increasing stretch magnitudes. Furthermore, we observed a first distinct inflection point in the force-elongation curve at an average sarcomere length of 3.5μm that was associated with an average force of 68±5nN/mm. This inflection point was thought to reflect the onset of Ig domain unfolding and was missing after a ten minute rest at zero force, suggesting a lack of spontaneous Ig domain refolding. These passive myofibrillar properties observed here are consistent with those observed in isolated titin molecules, suggesting that the mechanics of titin are well preserved in isolated myofibrils, and thus, can be studied readily in myofibrils, rather than in the extremely difficult and labile single titin preparations.     

Contributions of stretch activation to length-dependent contraction in murine myocardium

The steep relationship between systolic force production and end diastolic volume (Frank-Starling relationship) in myocardium is a potentially important mechanism by which the work capacity of the heart varies on a beat-to-beat basis, but the molecular basis for the effects of myocardial fiber length on cardiac work are still not well understood. Recent studies have suggested that an intrinsic property of myocardium, stretch activation, contributes to force generation during systolic ejection in myocardium. To examine the role of stretch activation in length dependence of activation we recorded the force responses of murine skinned myocardium to sudden stretches of 1% of muscle length at both short (1.90 microm) and long (2.25 microm) sarcomere lengths (SL). Maximal Ca(2+)-activated force and Ca(2+) sensitivity of force were greater at longer SL, such that more force was produced at a given Ca(2+) concentration. Sudden stretch of myocardium during an otherwise isometric contraction resulted in a concomitant increase in force that quickly decayed to a minimum and was followed by a delayed development of force, i.e., stretch activation, to levels greater than prestretch force. At both maximal and submaximal activations, increased SL significantly reduced the initial rate of force decay following stretch; at submaximal activations (but not at maximal) the rate of delayed force development was accelerated. This combination of mechanical effects of increased SL would be expected to increase force generation during systolic ejection in vivo and prolong the period of ejection. These results suggest that sarcomere length dependence of stretch activation contributes to the steepness of the Frank-Starling relationship in living myocardium.     

A non-cross-bridge stiffness in activated frog muscle fibers

Force responses to fast ramp stretches of various amplitude and velocity, applied during tetanic contractions, were measured in single intact fibers from frog tibialis anterior muscle. Experiments were performed at 14 degrees C at approximately 2.1 microm sarcomere length on fibers bathed in Ringer's solution containing various concentrations of 2,3-butanedione monoxime (BDM) to greatly reduce the isometric tension. The fast tension transient produced by the stretch was followed by a period, lasting until relaxation, during which the tension remained constant to a value that greatly exceeded the isometric tension. The excess of tension was termed "static tension," and the ratio between the force and the accompanying sarcomere length change was termed "static stiffness." The static stiffness was independent of the active tension developed by the fiber, and independent of stretch amplitude and stretching velocity in the whole range tested; it increased with sarcomere length in the range 2.1-2.8 microm, to decrease again at longer lengths. Static stiffness increased well ahead of tension during the tetanus rise, and fell ahead of tension during relaxation. These results suggest that activation increased the stiffness of some sarcomeric structure(s) outside the cross-bridges.     

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.     

The mechanisms of the residual force enhancement after stretch of skeletal muscle: non-uniformity in half-sarcomeres and stiffness of titin

When activated skeletal muscles are stretched, the force increases significantly. After the stretch, the force decreases and reaches a steady-state level that is higher than the force produced at the corresponding length during purely isometric contractions. This phenomenon, referred to as residual force enhancement, has been observed for more than 50 years, but the mechanism remains elusive, generating considerable debate in the literature. This paper reviews studies performed with single muscle fibres, myofibrils and sarcomeres to investigate the mechanisms of the stretch-induced force enhancement. First, the paper summarizes the characteristics of force enhancement and early hypotheses associated with non-uniformity of sarcomere length. Then, it reviews new evidence suggesting that force enhancement can also be associated with sarcomeric structures. Finally, this paper proposes that force enhancement is caused by: (i) half-sarcomere non-uniformities that will affect the levels of passive forces and overlap between myosin and actin filaments, and (ii) a Ca(2+)-induced stiffness of titin molecules. These mechanisms are compatible with most observations in the literature, and can be tested directly with emerging technologies in the near future.     

Titin determines the Frank-Starling relation in early diastole

Titin, a giant protein spanning half the sarcomere, is responsible for passive and restoring forces in cardiac myofilaments during sarcomere elongation and compression, respectively. In addition, titin has been implicated in the length-dependent activation that occurs in the stretched sarcomere, during the transition from diastole to systole. The purpose of this study was to investigate the role of titin in the length-dependent deactivation that occurs during early diastole, when the myocyte is shortened below slack length. We developed a novel in vitro assay to assess myocyte restoring force (RF) by measuring the velocity of recoil in Triton-permeabilized, unloaded rat cardiomyocytes after rigor-induced sarcomere length (SL) contractions. We compared rigor-induced SL shortening to that following calcium-induced (pCa) contractions. The RF-SL relationship was linearly correlated, and the SL-pCa curve displayed a characteristic sigmoidal curve. The role of titin was defined by treating myocytes with a low concentration of trypsin, which we show selectively degrades titin using mass spectroscopic analysis. Trypsin treatment reduced myocyte RF as shown by a decrease in the slope of the RF-SL relationship, and this was accompanied by a downward and leftward shift of the SL-pCa curve, indicative of sensitization of the myofilaments to calcium. In addition, trypsin digestion did not alter the relationship between SL and interfilament spacing (assessed by cell width) after calcium activation. These data suggest that as the sarcomere shortens below slack length, titin-based restoring forces act to desensitize the myofilaments. Furthermore, in contrast to length-dependent activation at long SLs, length-dependent deactivation does not depend on interfilament spacing. This study demonstrates for the first time the importance of titin-based restoring force in length-dependent deactivation during the early phase of diastole.     

Stretching molecular springs: elasticity of titin filaments in vertebrate striated muscle

Titin, the giant protein of striated muscle, provides a continuous link between the Z-disk and the M-line of a sarcomere. The elastic I-band section of titin comprises two main structural elements, stretches of immunoglobulin-like domains and a unique sequence, the PEVK segment. Both elements contribute to the extensibility and passive force development of nonactivated muscle. Extensibility of the titin segments in skeletal muscle has been determined by immunofluorescence/immunoelectron microscopy of sarcomeres stained with sequence-assigned titin antibodies. The force developed upon stretch of titin has been measured on isolated molecules or recombinant titin fragments with the help of optical tweezers and the atomic force microscope. Force has also been measured in single isolated myofibrils. The force-extension relation of titin could be readily fitted with models of biopolymer elasticity. For physiologically relevant extensions, the elasticity of the titin segments was largely explainable by an entropic-spring mechanism. The modelling explains why during stretch of titin, the Ig-domain regions (with folded modules) extend before the PEVK domain. In cardiac muscle, I-band titin is expressed in different isoforms, termed N2-A and N2-B. The N2-A isoform resembles that of skeletal muscle, whereas N2-B titin is shorter and is distinguished by cardiac-specific Ig-motifs and nonmodular sequences within the central I-band section. Examination of N2-B titin extensibility revealed that this isoform extends by recruiting three distinct elastic elements: poly-Ig regions and the PEVK domain at lower stretch and, in addition, a unique 572-residue sequence insertion at higher physiological stretch. Extension of all three elements allows cardiac titin to stretch fully reversibly at physiological sarcomere lengths, without the need to unfold individual Ig domains. However, unfolding of a very small number of Ig domains remains a possibility.     

Passive and active tension in single cardiac myofibrils.

Single myofibrils were isolated from chemically skinned rabbit heart and mounted in an apparatus described previously (Fearn et al., 1993; Linke et al., 1993). We measured the passive length-tension relation and active isometric force, both normalized to cross sectional area. Myofibrillar cross sectional area was calculated based on measurements of myofibril diameter from both phase-contrast images and electron micrographs. Passive tension values up to sarcomere lengths of approximately 2.2 microns were similar to those reported in larger cardiac muscle specimens. Thus, the element responsible for most, if not all, passive force of cardiac muscle at physiological sarcomere lengths appears to reside within the myofibrils. Above 2.2 microns, passive tension continued to rise, but not as steeply as reported in multicellular preparations. Apparently, structures other than the myofibrils become increasingly important in determining the magnitude of passive tension at these stretched lengths. Knowing the myofibrillar component of passive tension allowed us to infer the stress-strain relation of titin, the polypeptide thought to support passive force in the sarcomere. The elastic modulus of titin is 3.5 x 10(6) dyn cm-2, a value similar to that reported for elastin. Maximum active isometric tension in the single myofibril at sarcomere lengths of 2.1-2.3 microns was 145 +/- 35 mN/mm2 (mean +/- SD; n = 15). This value is comparable with that measured in fixed-end contractions of larger cardiac specimens, when the amount of nonmyofibrillar space in those preparations is considered. However, it is about 4 times lower than the maximum active tension previously measured in single skeletal myofibrils under similar conditions (Bartoo et al., 1993).     

Mechanics and structure of titin oligomers explored with atomic force microscopy

Titin is a giant polypeptide that spans half of the striated muscle sarcomere and generates passive force upon stretch. To explore the elastic response and structure of single molecules and oligomers of titin, we carried out molecular force spectroscopy and atomic force microscopy (AFM) on purified full-length skeletal-muscle titin. From the force data, apparent persistence lengths as long as approximately 1.5 nm were obtained for the single, unfolded titin molecule. Furthermore, data suggest that titin molecules may globally associate into oligomers which mechanically behave as independent wormlike chains (WLCs). Consistent with this, AFM of surface-adsorbed titin molecules revealed the presence of oligomers. Although oligomers may form globally via head-to-head association of titin, the constituent molecules otherwise appear independent from each other along their contour. Based on the global association but local independence of titin molecules, we discuss a mechanical model of the sarcomere in which titin molecules with different contour lengths, corresponding to different isoforms, are held in a lattice. The net force response of aligned titin molecules is determined by the persistence length of the tandemly arranged, different WLC components of the individual molecules, the ratio of their overall contour lengths, and by domain unfolding events. Biased domain unfolding in mechanically selected constituent molecules may serve as a compensatory mechanism for contour- and persistence-length differences. Variation in the ratio and contour length of the component chains may provide mechanisms for the fine-tuning of the sarcomeric passive force response.     

Mechanics and structure of titin oligomers explored with atomic force microscopy

Titin is a giant polypeptide that spans half of the striated muscle sarcomere and generates passive force upon stretch. To explore the elastic response and structure of single molecules and oligomers of titin, we carried out molecular force spectroscopy and atomic force microscopy (AFM) on purified full-length skeletal-muscle titin. From the force data, apparent persistence lengths as long as ∼1.5 nm were obtained for the single, unfolded titin molecule. Furthermore, data suggest that titin molecules may globally associate into oligomers which mechanically behave as independent wormlike chains (WLCs). Consistent with this, AFM of surface-adsorbed titin molecules revealed the presence of oligomers. Although oligomers may form globally via head-to-head association of titin, the constituent molecules otherwise appear independent from each other along their contour. Based on the global association but local independence of titin molecules, we discuss a mechanical model of the sarcomere in which titin molecules with different contour lengths, corresponding to different isoforms, are held in a lattice. The net force response of aligned titin molecules is determined by the persistence length of the tandemly arranged, different WLC components of the individual molecules, the ratio of their overall contour lengths, and by domain unfolding events. Biased domain unfolding in mechanically selected constituent molecules may serve as a compensatory mechanism for contour- and persistence-length differences. Variation in the ratio and contour length of the component chains may provide mechanisms for the fine-tuning of the sarcomeric passive force response.     

Neutrophil accumulation following passive stretches contributes to adaptations that reduce contraction-induced skeletal muscle injury in mice.

Skeletal muscles can be injured by their own contractions, especially when the muscle is stretched during a lengthening contraction. Exposing a muscle to a conditioning protocol of stretches without activation (passive stretches) before lengthening contractions reduces contraction-induced injury. Although passive stretching does not damage muscle fibers, neutrophils are elevated in the muscle after passive stretches. Our purpose was to investigate the relationship between neutrophil accumulation following passive stretches and the protection from subsequent contraction-induced injury provided by the passive stretches. Our hypothesis was that passive stretch conditioning would not provide protection from subsequent lengthening contraction-induced injury under circumstances when the increase in muscle neutrophils in response to the conditioning was prevented. Extensor digitorum longus muscles of mice were conditioned with passive stretches 14 days before exposure to a protocol of damaging lengthening contractions. Mice were either untreated or treated with an antibody (RB6-8C5) that reduced the level of circulating neutrophils by over 95% before administration of passive stretches. Neutrophil levels recovered in treated mice by the time lengthening contractions were performed. Lengthening contractions were also administered to muscles with no prior exposure to passive stretches. Maximum isometric force, number of damaged fibers, and muscle neutrophil concentration were measured 3 days after lengthening contractions. Compared with nonconditioned control muscles, the severity of contraction-induced injury was not reduced by prior passive stretch conditioning when mice were treated with RB6-8C5 before conditioning. We conclude that neutrophils contribute to adaptations that protect muscles from injury.