Gaining new understanding of sarcomere length non-uniformities in skeletal muscles.

Sarcomere lengths are non-uniform on all structural levels of mammalian skeletal muscle. These non-uniformities have been associated with a variety of mechanical properties, including residual force enhancement and depression, creep, increased force capacity, and extension of the plateau of the force-length relationship. However, the nature of sarcomere length non-uniformities has not been explored systematically. The purpose of this study was to determine the properties of sarcomere length non-uniformities in active and passive muscle. Single myofibrils of rabbit psoas (n = 20; with 412 individual sarcomeres) were subjected to three activation/deactivation cycles and individual sarcomere lengths were measured at 4 passive and 3 active points during the activation/deactivation cycles. The myofibrils were divided into three groups based on their initial average sarcomere lengths: short, intermediate, and long average sarcomere lengths of 2.7, 3.2, and 3.6 µm. The primary results were that sarcomere length non-uniformities did not occur randomly but were governed by some structural and/or contractile properties of the sarcomeres and that sarcomere length non-uniformities increased when myofibrils went from the passive to the active state. We propose that the mechanisms that govern the systematic sarcomere lengths non-uniformities observed in active and passive myofibrils may be associated with the variable number of contractile proteins and the variable number and the adjustable stiffness of titin filaments in individual sarcomeres.

Residual Force Enhancement Following Eccentric Contractions: A New Mechanism Involving Titin.

Eccentric muscle properties are not well characterized by the current paradigm of the molecular mechanism of contraction: the cross-bridge theory. Findings of force contributions by passive structural elements a decade ago paved the way for a new theory. Here, we present experimental evidence and theoretical support for the idea that the structural protein titin contributes to active force production, thereby explaining many of the unresolved properties of eccentric muscle contraction.

Using diet-induced obesity to understand a metabolic subtype of osteoarthritis in rats.

UNLABELLED: Osteoarthritis (OA) in obese individuals is often attributed to joint loading. However, a subtype of OA, Metabolic OA, may be due to obesity-related intrinsic factors but remains to be evaluated experimentally against a known OA progression model. OBJECTIVE: To evaluate if obesity contributes to OA onset using a high fat/high sucrose diet-induced obesity (DIO) model with anterior cruciate ligament-transected rats (ACL-X). METHODS: Sprague Dawley rats (n = 33) consumed high fat/high sucrose or chow diets for 12 weeks, were randomized to one of three groups: a unilateral ACL-X group, sham surgery group, or naïve non-surgical group. These animals were followed for an additional 16 weeks. At sacrifice, body composition, knee joint Modified Mankin scores, and 27 serum and synovial fluid cytokines and adipokines were measured. RESULTS: Experimental limbs of obese ACL-X, obese Sham, and lean ACL-X animals had similar Modified Mankin scores that were greater than those obtained from lean Sham and naïve animals. Obese contralateral limbs had similar OA damage as ACL-X and Sham limbs of obese and ACL-X limbs of lean animals. Obese contralateral limb Modified Mankin scores had a strong correlation (r = 0.75, P < 0.001) with body fat percentage. Serum leptin and synovial fluid IP10/CXCL10 best described Modified Mankin scores in contralateral limbs of obese animals. CONCLUSIONS: Mechanical factors produced OA damage in experimental limbs, as expected. Interestingly, OA damage in obese contralateral limbs was similar to mechanically perturbed limbs, suggesting that obesity may induce OA in a non-mechanical manner.

Titin (visco-) elasticity in skeletal muscle myofibrils.

Titin is the third most abundant protein in sarcomeres and fulfills a number of mechanical and signaling functions. Specifically, titin is responsible for most of the passive forces in sarcomeres and the passive visco-elastic behaviour of myofibrils and muscles. It has been suggested, based on mechanical testing of isolated titin molecules, that titin is an essentially elastic spring if Ig domain un/refolding is prevented either by working at short titin lengths, prior to any un- folding of Ig domains, or at long sarcomere (and titin) lengths when Ig domain un/refolding is effectively prevented. However, these properties of titin, and by extension of muscles, have not been tested with titin in its natural structural environment within a sarcomere. The purpose of this study was to gain insight into the Ig domain un/refolding kinetics and test the idea that titin could behave essentially elastically at any sarcomere length by preventing Ig domain un/refolding during passive stretch-shortening cycles. Although not completely successful, we demonstrate here that titin's visco-elastic properties appear to depend on the Ig do- main un/refolding kinetics and that indeed, titin (and thus myofibrils) can become virtually elastic when Ig domain un/refolding is prevented.

Vertebral artery strains during high-speed, low amplitude cervical spinal manipulation.

Spinal manipulative therapy (SMT) has been recognized as an effective treatment modality for many back, neck and musculoskeletal problems. One of the major issues of the use of SMT is its safety, especially with regards to neck manipulation and the risk of stroke. The vast majority of these accidents involve the vertebro-basilar system, specifically the vertebral artery (VA) between C2/C1. However, the mechanics of this region of the VA during SMT are unexplored. Here, we present first ever data on the mechanics of this region during cervical SMT performed by clinicians. VA strains obtained during SMT are significantly smaller than those obtained during diagnostic and range of motion testing, and are much smaller than failure strains. We conclude from this work that cervical SMT performed by trained clinicians does not appear to place undue strain on VA, and thus does not seem to be a factor in vertebro-basilar injuries.

The force-length relationship of mechanically isolated sarcomeres.

The sarcomere force-length relationship is arguably the most basic property of skeletal muscle force production. It has been accepted as textbook knowledge and is in direct support of the sliding filament and cross-bridge theories of contraction. However, the sarcomere force-length relationship has never been measured directly. Here, we show results of two experiments elucidating the force-length properties of mechanically isolated sarcomeres. We demonstrate that sarcomere forces are greatly dependent on sarcomere lengths for purely isometric conditions, but can take on essentially any steady-state value depending on an individual sarcomere's contractile history. Therefore, we conclude that steady-state isometric forces in isolated sarcomeres do not only depend on sarcomere lengths (or equivalently actin-myosin overlap) but depend crucially on a sarcomere's contractile history. These results have direct implications for our understanding of the molecular mechanisms of muscle contraction.

Force enhancement following stretch in a single sarcomere.

It has been accepted for half a century that, for a given level of activation, the steady-state isometric force of a muscle sarcomere depends exclusively on the amount of overlap between the contractile filaments actin and myosin, or equivalently sarcomere length (Gordon AM et al., J Physiol 184: 170-192, 1966). Moreover, according to the generally accepted paradigm of muscle contraction, the cross-bridge theory (Huxley AF, Prog Biophys Biophys Chem 7: 255-318, 1957), this steady-state isometric sarcomere force is independent of the muscle's contractile history (Huxley AF, Prog Biophys Biophys Chem 7: 255-318, 1957; Walcott S and Herzog W, Math Biosci 216: 172-186, 2008); i.e., it is independent of whether a muscle is held at a constant length before and during the contraction or whether the muscle is shortened or lengthened to the same constant length. This, however, is not the case, as muscles and single fibers that are stretched show greatly increased steady-state isometric forces compared with preparations that are held at a constant length (Abbott BC and Aubert XM, J Physiol 117: 77-86, 1952; De Ruiter CJ et al., J Physiol 526.3: 671-681, 2000; Edman KAP et al., J Physiol 281: 139-155, 1978; Edman KAP et al., J Gen Physiol 80: 769-784, 1982; Edman KAP and Tsuchiya T, J Physiol 490.1: 191-205, 1996). This so-called "residual force enhancement" (Edman KAP et al., J Gen Physiol 80: 769-784, 1982) offers a perplexing puzzle for muscle physiologists. Many theories have been advanced to address the discrepancy between prediction and observation with the most popular and accepted being the sarcomere length nonuniformity theory (Morgan DL, Biophys J 57: 209-221, 1990), which explains the residual force enhancement with the development of large nonuniformities in sarcomere lengths during muscle stretching. Here, we performed experiments in mechanically isolated sarcomeres and observed that the residual force enhancement following active stretching is preserved. Since our preparation utilizes a single sarcomere, a redistribution of the length of neighboring sarcomeres to produce the higher force following stretch is, by design, precluded. Furthermore, the enhanced forces in the single sarcomeres always exceed the isometric forces on the plateau of the force-length relationship, thereby eliminating the possibility that our result might have been obtained because of a redistribution of half-sarcomere lengths. Since force enhancement in single myofibrils has been associated with actin-titin interactions (Kulke M et al., Circ Res 89: 874-881, 2001; Li Q et al., Biophys J 69: 1508-1518, 1995) and calcium binding to titin (Joumaa V et al., Am J Physiol Cell Physiol 294: C74-C78, 2008; Labeit D et al., Proc Natl Acad Sci USA 100: 13716-13721, 2003), titin may regulate the sarcomeric force enhancement observed here.

An activatable molecular spring reduces muscle tearing during extreme stretching.

The purpose of this study was to determine failure stresses and failure lengths of actively and passively stretched myofibrils. As expected, myofibrils failed at average sarcomere lengths (about 6-7µm) that vastly exceeded sarcomere lengths at which actin-myosin filament overlap ceases to exist (4µm) and thus actin-myosin-based cross-bridge forces are zero at failure. Surprisingly, however, actively stretched myofibrils had much greater failure stresses and failure energies than passively stretched myofibrils, thereby providing compelling evidence for strong force production independent of actin-myosin-based cross-bridge forces. Follow-up experiments in which titin was deleted and cross-bridge formation was inhibited at high and low calcium concentrations point to titin as the regulator of this force, independent of calcium. The results of this study point to a mechanism of force production that reduces stretch-induced muscle damage at extreme length and limits injury and force loss within physiologically relevant ranges of sarcomere and muscle lengths.

Regulation of muscle force in the absence of actin-myosin-based cross-bridge interaction.

For the past half century, the sliding filament-based cross-bridge theory has been the cornerstone of our understanding of how muscles contract. According to this theory, active force can only occur if there is overlap between the contractile filaments, actin and myosin. Otherwise, forces are thought to be caused by passive structural elements and are assumed to vary solely because of the length of the muscle. We observed increases in muscle force by a factor of 3 to 4 above the purely passive forces for activated and stretched myofibrils in the absence of actin-myosin overlap. We show that this dramatic increase in force is crucially dependent on the presence of the structural protein titin, cannot be explained with calcium activation, and is regulated by actin-myosin-based cross-bridge forces before stretching. We conclude from these observations that titin is a strong regulator of muscle force and propose that this regulation is based on cross-bridge force-dependent titin-actin interactions. These results suggest a mechanism for stability of sarcomeres on the "inherently unstable" descending limb of the force-length relationship, and they further provide an explanation for the protection of muscles against stretch-induced muscle injuries.

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.

The origin of passive force enhancement in skeletal muscle.

The aim of the present study was to test whether titin is a calcium-dependent spring and whether it is the source of the passive force enhancement observed in muscle and single fiber preparations. We measured passive force enhancement in troponin C (TnC)-depleted myofibrils in which active force production was completely eliminated. The TnC-depleted construct allowed for the investigation of the effect of calcium concentration on passive force, without the confounding effects of actin-myosin cross-bridge formation and active force production. Passive forces in TnC-depleted myofibrils (n = 6) were 35.0 +/- 2.9 nN/ microm(2) when stretched to an average sarcomere length of 3.4 microm in a solution with low calcium concentration (pCa 8.0). Passive forces in the same myofibrils increased by 25% to 30% when stretches were performed in a solution with high calcium concentration (pCa 3.5). Since it is well accepted that titin is the primary source for passive force in rabbit psoas myofibrils and since the increase in passive force in TnC-depleted myofibrils was abolished after trypsin treatment, our results suggest that increasing calcium concentration is associated with increased titin stiffness. However, this calcium-induced titin stiffness accounted for only approximately 25% of the passive force enhancement observed in intact myofibrils. Therefore, approximately 75% of the normally occurring passive force enhancement remains unexplained. The findings of the present study suggest that passive force enhancement is partly caused by a calcium-induced increase in titin stiffness but also requires cross-bridge formation and/or active force production for full manifestation.

Passive force enhancement in single myofibrils.

The purpose of this study was to gain further insight into passive force enhancement by testing whether passive force enhancement occurs in single myofibrils. Myofibrils (n = 6) isolated from rabbit psoas muscle were fixed at a sarcomere length of 2.4 microm, and then stretched passively and actively to a sarcomere length of 3.4 microm. Passive force after deactivation of the myofibrils was increased after active compared to passive stretching. Therefore, passive force enhancement, previously observed in muscle and fiber preparations, also occurs in single myofibrils. Passive force enhancement in myofibrils ranged from 86 to 145% of the steady-state force observed after passive stretch. Because titin is the main source of passive force in myofibrils, we propose that titin might be responsible for passive force enhancement observed in myofibrils. We propose that this might occur through an increase in stiffness when calcium concentration increases upon activation.

Residual force depression is not abolished following a quick shortening step.

Residual force depression is long lasting, but can be abolished instantaneously when a muscle is deactivated just long enough for force to drop to zero. According to the "cross-bridge inhibition theory" of force depression, this result is predicted as the release of stress on actin during deactivation restores the angular distortion of the actin binding sites, thereby establishing conditions identical to those of a purely isometric contraction. According to this theory, force depression should also be abolished if stress on actin is released through a quick shortening step. For slow (4.5mm/s) shortening of cat soleus (n=8), force depression was achieved in all muscles and averaged 5.3% (+/-1.9%) and 5.8% (+/-1.3%) for 9 and 18 mm shortening amplitudes. Following quick shortening (200 mm/s) of 18 mm, there was no statistically significant force depression. However, when slow shortening (4.5mm/s for 9 mm) was followed by quick shortening (200 mm/s for 9 mm) after delays of 0, 1, and 2s, there was a small but significant force depression in all cases averaging 3.2%, 3.7%, and 4.2%, respectively. We conclude from these results that a small amount of force depression persists following stress release caused by quick shortening, and therefore that the cross-bridge inhibition theory cannot be the sole cause of force depression.

Heterogeneity in patellofemoral cartilage adaptation to anterior cruciate ligament transection; chondrocyte shape and deformation with compression.

OBJECTIVE: The purpose of this study was to determine if the opposing cartilages of the feline patellofemoral joint adapted differently to short-term anterior cruciate ligament transection (ACL-T) and if the magnitude of chondrocyte deformation upon tissue loading was altered under ACL-T conditions compared to contralateral controls. In situ static compression of physiological magnitude was applied to the feline patellofemoral cartilage 16 weeks post-ACL-T and cartilage and chondrocyte deformation were evaluated by histomorphometry. DESIGN: Six adult cats were euthanized 16 weeks after unilateral ACL-T. A peak surface pressure of 9 MPa was applied to the fully intact patella and femoral groove cartilages. After in situ fixation under compression, sections from the centre of the indent and from an adjacent unloaded area of the cartilages were analysed. Chondrocyte shape, size, clustering and volumetric fraction were quantified. RESULTS: Experimental patellar articular cartilage was thicker, contained larger chondrocytes that were more frequently arranged in clusters and had, on average, a larger chondrocyte volumetric fraction compared to contralateral controls. In contrast, the experimental femoral groove cartilage demonstrated little adaptation to ACL-T. CONCLUSIONS: The patellar articular cartilage adapts to short-term ACL-T to a greater extent than femoral groove cartilage. We speculate that differences in the histological parameters of control tissues, such as cartilage thickness and the magnitude and depth distribution of chondrocyte shape, size and volumetric fraction may contribute to predisposing patellar cartilage, and not femoral groove cartilage, to adaptation after ACL-T.

Opposing cartilages in the patellofemoral joint adapt differently to long-term cruciate deficiency: chondrocyte deformation and reorientation with compression.

OBJECTIVE: The purposes of this study were to quantify patellofemoral histology in the feline knee 67 months post-anterior cruciate ligament transection (ACL-T) and to apply an in situ static load of physiological magnitude to the articular cartilage and evaluate the resulting cartilage and chondrocyte deformation. DESIGN: Six cats were sacrificed 67+/-6 months post-unilateral ACL-T. Static compression was applied to the cartilage surfaces of the patellofemoral joint using a cylindrical metal indentor. After fixation, full thickness osteochondral blocks were harvested and sections cut from not-indented and indented areas. Chondrocyte shape, orientation and volumetric fraction as well as cartilage thickness were evaluated. RESULTS: Experimental and contralateral patellae were histologically different compared to normal with thickened cartilage, rounded superficial chondrocytes, and uneven proteoglycan staining throughout. In contrast, no differences were apparent in 10 of the 12 femoral groove samples. The structural reorganisation of the experimental patellae cartilage that occurred with load was also different compared to normal. Specifically, the indentation shape was deeper and had steeper sides and the realignment of deep zone cells at angles of 45 degrees and 135 degrees observed in normal cartilage was no longer apparent in the experimental tissue. CONCLUSIONS: Two directly articulating cartilage surfaces of the feline patellofemoral joint have completely contrasting responses to long-term ACL-T. We speculate that this could be a result of the different nature of the loads experienced by the two surfaces (intermittent vs constant) and/or the differences in the histology and material properties of the two tissues in their normal state, and/or an inherent difference in the biological response capabilities of the articular cartilages.

Does the speed of shortening affect steady-state force depression in cat soleus muscle?

It has been stated repeatedly for the past 50 years that the steady-state force depression following shortening of an activated muscle depends on the speed of shortening. However, these statements were based on results from experiments in which muscles were shortened at different speeds but identical activation levels. Therefore, the force during shortening was changed in accordance with the force-velocity relationship of muscles: that is, increasing speeds of shortening were associated with decreasing forces, and vice versa. Consequently, it is not possible at present to distinguish whether force depression is caused by the changes in speed, as frequently stated, or the associated changes in force, or both. The purpose of this study was to test if force depression depends on the speed of shortening. We hypothesized that force depression was dependent on the force but not the speed of contraction. Our prediction is that the amount of force depression after shortening contractions at different speeds could be similar if the force during contraction was controlled at a similar level. Cat soleus muscles (n=7) were shortened by 9 or 12 mm at speeds of 3, 9, and 27 mm/s, first with a constant activation during shortening (30Hz), then with activation levels that were reduced (<30Hz) for the slow speeds (3 and 9 mm/s) to approximate the shortening forces of the fast speed contractions (27 mm/s). If done properly, force depression could be precisely matched at the three different speeds, indicating that force depression was related to the force during the shortening contraction but not to the speed. However, in order to match force depression, the forces during shortening had to be systematically greater for the slow compared to the fast speeds of shortening, suggesting that force depression also depends on the level of activation, as force depression at constant activation levels can only be matched if the force during shortening, evaluated by the mechanical work, is identical. Therefore, we conclude that force depression depends on the force and activation level during shortening, but does not depend on the speed of shortening as has been assumed for half a century. These results support, but do not prove, the current hypothesis that force depression is caused by a stress-related cross-bridge inhibition in the actin-myosin overlap zone that is newly formed during muscle shortening.

Proposed model of botulinum toxin-induced muscle weakness in the rabbit.

Osteoarthritic patients show only a weak association between radiographic signs of joint disease and joint pain and disability. Conversely, muscle weakness is one of the earliest and most common symptoms of patients with osteoarthritis (OA). However, while many experimental models of osteoarthritis include a component of muscular weakness, no model has isolated this factor satisfactorily. Therefore, the purpose of this study was to develop and validate an experimental animal model of muscle weakness for future use in the study of OA. Botulinum Type-A toxin (BTX-A) was uni-laterally injected into the quadriceps musculature of New Zealand white rabbits (3.5 units/kg). Isometric knee extensor torque at a range of knee angles and stimulation frequencies, and quadriceps muscle mass, were quantified for control animals, and at one- and six-months post-repeated injections, in both, the experimental and the contralateral hindlimb. Ground reaction forces were measured in all animals while hopping across two force platforms. Isometric knee extension torque and quadriceps muscle mass was systematically decreased in the experimental hindlimb. Vertical ground reaction forces in the push off phase of hopping were also decreased in the experimental compared to control hindlimbs. We conclude that BTX-A injection into the rabbit musculature creates functional and absolute muscle weakness in a reproducible manner. Therefore, this model may be used to systematically study the possible effects of muscle weakness on joint degeneration, either as an isolated intervention, or in combination with other interventions (anterior cruciate ligament transection, meniscectomy) known to create knee joint degeneration.

The role of passive structures in force enhancement of skeletal muscles following active stretch.

We recently found that force enhancement following active stretch in skeletal muscles is accompanied by an increase in passive force following deactivation (J. Exp. Biol. 205 (2002) 1275). However, it is not known if this increase in passive force contributes to the force enhancement observed in the active muscle, and if it is observed at all muscle lengths. The purposes of this study were to quantify the amount of passive force increase as a function of muscle lengths, and to determine if this passive force contributes to the force enhancement observed in the active muscle. Experiments were performed on cat soleus (n = 24) using techniques published previously (J. Biomech. 30(9) (1997) 865). Conceptually, tests involved comparisons of force enhancement and passive force increase for a variety of stretch tests in soleus. Furthermore, in one test, activation of the soleus was interrupted for 1s in the force-enhanced state, and soleus was then re-activated. We found that total force enhancement and passive force increase were positively correlated for all test conditions, that passive force increase following stretch of the active soleus only occurred at muscle lengths corresponding to the descending limb of the force-length relationship, that increases in passive force for a given stretch magnitude became greater at long muscle lengths, and that upon reactivation, there was a remnant passive force enhancement. We conclude from these results that the passive force enhancement following stretch of an active muscle contributes to the total force enhancement, that this passive contribution increases with increasing muscle length, and that there must be at least one other factor than passive force increase that contributes to the total force enhancement, as the passive force increase was always smaller than the total force enhancement. A by-product of this investigation was that we observed a shift in the passive force-length relationship that was dependent on muscle activation, stretch magnitude and muscle length. Therefore, the passive force-length relationship is not a constant property of skeletal muscle, but depends critically on the muscle's contractile history.

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.