The force response of muscles to activation and length perturbations depends on length history.

Recent studies have demonstrated that muscle force is not determined solely by activation under dynamic conditions, and that length history has an important role in determining dynamic muscle force. Yet, the mechanisms for how muscle force is produced under dynamic conditions remain unclear. To explore this, we investigated the effects of muscle stiffness, activation and length perturbations on muscle force. First, submaximal isometric contraction was established for whole soleus muscles. Next, the muscles were actively shortened at three velocities. During active shortening, we measured muscle stiffness at optimal muscle length (L0) and the force response to time-varying activation and length perturbations. We found that muscle stiffness increased with activation but decreased as shortening velocity increased. The slope of the relationship between maximum force and activation amplitude differed significantly among shortening velocities. Also, the intercept and slope of the relationship between length perturbation amplitude and maximum force decreased with shortening velocity. As shortening velocities were related to muscle stiffness, the results suggest that length history determines muscle stiffness and the history-dependent muscle stiffness influences the contribution of activation and length perturbations to muscle force. A two-parameter viscoelastic model including a linear spring and a linear damper in parallel with measured stiffness predicted history-dependent muscle force with high accuracy. The results and simulations support the hypothesis that muscle force under dynamic conditions can be accurately predicted as the force response of a history-dependent viscoelastic material to length perturbations.

Understanding muscle function during perturbed in vivo locomotion using a muscle avatar approach.

The work loop technique has provided key insights into in vivo muscle work and power during steady locomotion. However, for many animals and muscles, ex vivo experiments are not feasible. In addition, purely sinusoidal strain trajectories lack variations in strain rate that result from variable loading during locomotion. Therefore, it is useful to develop an 'avatar' approach in which in vivo strain and activation patterns from one muscle are replicated in ex vivo experiments on a readily available muscle from an established animal model. In the present study, we used mouse extensor digitorum longus (EDL) muscles in ex vivo experiments to investigate in vivo mechanics of the guinea fowl lateral gastrocnemius (LG) muscle during unsteady running on a treadmill with obstacle perturbations. In vivo strain trajectories from strides down from obstacle to treadmill, up from treadmill to obstacle, strides with no obstacle and sinusoidal strain trajectories at the same amplitude and frequency were used as inputs in work loop experiments. As expected, EDL forces produced with in vivo strain trajectories were more similar to in vivo LG forces (R2=0.58-0.94) than were forces produced with the sinusoidal trajectory (average R2=0.045). Given the same stimulation, in vivo strain trajectories produced work loops that showed a shift in function from more positive work during strides up from treadmill to obstacle to less positive work in strides down from obstacle to treadmill. Stimulation, strain trajectory and their interaction had significant effects on all work loop variables, with the interaction having the largest effect on peak force and work per cycle. These results support the theory that muscle is an active material whose viscoelastic properties are tuned by activation, and which produces forces in response to deformations of length associated with time-varying loads.

Transcriptomic profiles of muscular dystrophy with myositis (mdm) in extensor digitorum longus, psoas, and soleus muscles from mice.

BACKGROUND: Titinopathies are inherited muscular diseases triggered by genetic mutations in the titin gene. Muscular dystrophy with myositis (mdm) is one such disease caused by a LINE repeat insertion, leading to exon skipping and an 83-amino acid residue deletion in the N2A-PEVK region of mouse titin. This region has been implicated in a number of titin-titin ligand interactions, hence are important for myocyte signaling and health. Mice with this mdm mutation develop a severe and progressive muscle degeneration. The range of phenotypic differences observed in mdm mice shows that the deletion of this region induces a cascade of transcriptional changes extending to numerous signaling pathways affected by the titin filament. Previous research has focused on correlating phenotypic differences with muscle function in mdm mice. These studies have provided understanding of the downstream physiological effects resulting from the mdm mutation but only provide insights on processes that can be physiologically observed and measured. We used differential gene expression (DGE) to compare the transcriptomes of extensor digitorum longus (EDL), psoas and soleus muscles from wild-type and mdm mice to develop a deeper understand of these tissue-specific responses. RESULTS: The overall expression pattern observed shows a well-differentiated transcriptional signature in mdm muscles compared to wild type. Muscle-specific clusters observed within the mdm transcriptome highlight the level of variability of each muscle to the deletion. Differential gene expression and weighted gene co-expression network analysis showed a strong directional response in oxidative respiration-associated mitochondrial genes, which aligns with the poor shivering and non-shivering thermogenesis previously observed. Sln, which is a marker associated with shivering and non-shivering thermogenesis, showed the strongest expression change in fast-fibered muscles. No drastic changes in MYH expression levels were reported, which indicated an absence of major fiber-type switching events. Overall expression shifts in MYH isoforms, MARPs, and extracellular matrix associated genes demonstrated the transcriptional complexity associated with mdm mutation. The expression alterations in mitochondrial respiration and metabolism related genes in the mdm muscle dominated over other transcriptomic changes, and likely account for the late stage cellular responses in the mdm muscles. CONCLUSIONS: We were able to demonstrate that the complex nature of mdm mutation extends beyond a simple rearrangement in titin gene. EDL, psoas and soleus exemplify unique response modes observed in skeletal muscles with mdm mutation. Our data also raises the possibility that failure to maintain proper energy homeostasis in mdm muscles may contribute to the pathogenesis of the degenerative phenotype in mdm mice. Understanding the full disease-causing molecular cascade is difficult using bulk RNA sequencing techniques due to intricate nature of the disease. The development of the mdm phenotype is temporally and spatially regulated, hence future studies should focus on single fiber level investigations.

Residual force enhancement is reduced in permeabilized fiber bundles from mdm muscles.

Residual force enhancement (RFE) is the increase in steady-state force after active stretch relative to the force during isometric contraction at the same final length. The muscular dystrophy with myositis (mdm) mutation in mice, characterized by a small deletion in N2A titin, has been proposed to prevent N2A titin-actin interactions so that active mdm muscles are more compliant than wild type (WT). This decrease in active muscle stiffness is associated with reduced RFE. We investigated RFE in permeabilized soleus (SOL) and extensor digitorum longus (EDL) fiber bundles from WT and mdm mice. On each fiber bundle, we performed active and passive stretches from an average sarcomere length of 2.6-3.0 µm at a slow rate of 0.04 µm s-1, as well as isometric contractions at the initial and final lengths. One-way ANOVA showed that SOL and EDL fiber bundles from mdm mice exhibited significantly lower RFE than WT mice (P<0.0001). This result is consistent with previous observations in single myofibrils and intact muscles. However, it contradicts the results from a previous study that appeared to show that compensatory mechanisms could restore titin force enhancement in single fibers from mdm psoas. We suggest that RFE measured previously in mdm single fibers was an artifact of the high variability in passive tension found in degenerating fibers, which begins after ∼24 days of age. The results are consistent with the hypothesis that RFE is reduced in mdm skeletal muscles owing to impaired Ca2+-dependent titin-actin interactions resulting from the small deletion in N2A titin.

Micro-biopsies: a less invasive technique for investigating human muscle fiber mechanics.

The purpose of this investigation was to demonstrate that muscle fiber mechanics can be assessed on micro-biopsies obtained from human medial gastrocnemii. Three micro-biopsy samples were collected from female dancers (n=15). Single fibers and fiber bundles were isolated and passively stretched from 2.4 to 3.0 µm at 0.015 and 0.04 µm s-1 (n=50 fibers total) and in five increments at 0.12 µm s-1 (n=42 fibers total). Muscle fibers were then activated isometrically at 2.4 µm (n=4 fibers total) and 3.0 µm (n=3 fibers total). Peak stress and steady-state stress were significantly greater (P<0.0001) after stretching at 0.04 µm s-1 than at 0.015 µm s-1. Furthermore, peak stresses and steady-state stresses increased non-linearly with fiber length (P<0.0001). We conclude that active and passive muscle fiber mechanics can be investigated using tissue from micro-biopsies.

Contributions of Titin and Collagen to Passive Stress in Muscles from mdm Mice with a Small Deletion in Titin's Molecular Spring.

Muscular dystrophy with myositis (mdm) is a naturally occurring mutation in the mouse Ttn gene that results in higher passive stress in muscle fibers and intact muscles compared to wild-type (WT). The goal of this study was to test whether alternative splicing of titin exons occurs in mdm muscles, which contain a small deletion in the N2A-PEVK regions of titin, and to test whether splicing changes are associated with an increase in titin-based passive tension. Although higher levels of collagen have been reported previously in mdm muscles, here we demonstrate alternative splicing of titin in mdm skeletal muscle fibers. We identified Z-band, PEVK, and C-terminus Mex5 exons as splicing hotspots in mdm titin using RNA sequencing data and further reported upregulation in ECM-associated genes. We also treated skinned mdm soleus fiber bundles with trypsin, trypsin + KCl, and trypsin + KCL + KI to degrade titin. The results showed that passive stress dropped significantly more after trypsin treatment in mdm fibers (11 ± 1.6 mN/mm2) than in WT fibers (4.8 ± 1 mN/mm2; p = 0.0004). The finding that treatment with trypsin reduces titin-based passive tension more in mdm than in WT fibers supports the hypothesis that exon splicing leads to the expression of a stiffer and shorter titin isoform in mdm fibers. After titin extraction by trypsin + KCl + KI, mdm fibers (6.7 ± 1.27 mN/mm2) had significantly higher collagen-based passive stress remaining than WT fibers (2.6 ± 1.3 mN/mm2; p = 0.0014). We conclude that both titin and collagen contribute to higher passive tension of mdm muscles.

Titin: A Tunable Spring in Active Muscle.

Muscle has conventionally been viewed as a motor that converts chemical to kinetic energy in series with a passive spring, but new insights emerge when muscle is viewed as a composite material whose elastic elements are tuned by activation. New evidence demonstrates that calcium-dependent binding of N2A titin to actin increases titin stiffness in active skeletal muscles, which explains many long-standing enigmas of muscle physiology.

Muscle as a tunable material: implications for achieving muscle-like function in robotic prosthetic devices.

An ideal prosthesis should perform as well as or better than the missing limb it was designed to replace. Although this ideal is currently unattainable, recent advances in design have significantly improved the function of prosthetic devices. For the lower extremity, both passive prostheses (which provide no added power) and active prostheses (which add propulsive power) aim to emulate the dynamic function of the ankle joint, whose adaptive, time-varying resistance to applied forces is essential for walking and running. Passive prostheses fail to normalize energetics because they lack variable ankle impedance that is actively controlled within each gait cycle. By contrast, robotic prostheses can normalize energetics for some users under some conditions. However, the problem of adaptive and versatile control remains a significant issue. Current prosthesis-control algorithms fail to adapt to changes in gait required for walking on level ground at different speeds or on ramps and stairs. A new paradigm of 'muscle as a tunable material' versus 'muscle as a motor' offers insights into the adaptability and versatility of biological muscles, which may provide inspiration for prosthesis design and control. In this new paradigm, neural activation tunes muscle stiffness and damping, adapting the response to applied forces rather than instructing the timing and amplitude of muscle force. A mechanistic understanding of muscle function is incomplete and would benefit from collaboration between biologists and engineers. An improved understanding of the adaptability of muscle may yield better models as well as inspiration for developing prostheses that equal or surpass the functional capabilities of biological limbs across a wide range of conditions.

Huxleys' Missing Filament: Form and Function of Titin in Vertebrate Striated Muscle.

Although superthin filaments were inferred from early experiments on muscle, decades passed before their existence was accepted. Phylogenetic analyses suggest that titin, the largest known protein, first appeared in the common ancestor of chordates and nematodes and evolved rapidly via duplication. Twitchin and projectin evolved later by truncation. Sallimus mutants in Drosophila exhibit disrupted sarcomere and chromosome structure, suggesting that giant proteins may have evolved as chromosomal scaffolds that were co-opted for a similar purpose in striated muscles. Though encoded by only one gene, titin comprises hundreds of exons and has the potential for enormous diversity. Shorter isoforms typically confer greater passive stiffness associated with smaller in vivo muscle strains. Recent studies demonstrate unequivocally that titin stiffness increases upon muscle activation, but the mechanisms are only now being uncovered. Although some basic principles have been established, a vast opportunity remains to extend our understanding of titin function in striated muscle.

Stretch-Shortening Cycle Performance and Muscle-Tendon Properties in Dancers and Runners.

The purpose of this investigation was to elucidate whether ankle joint stretch-shortening cycle performance, isometric and isokinetic plantarflexion strength, and maximal Achilles tendon force and elongation differ between dancers, endurance runners, and untrained controls. To differentiate between dancers, endurance runners, and controls, the authors measured maximal Achilles tendon force and elongation during isometric ramp contractions with ultrasonic imaging, maximal isometric and isokinetic plantarflexion strength with dynamometry, and stretch-shortening cycle function during countermovement hopping and 30-cm drop hopping with a custom-designed sled. The Achilles tendon of dancers elongated significantly (P ≤ .05) more than runners and controls. Dancers were significantly stronger than controls during isometric contractions at different ankle angles. Concentric and eccentric strength during isokinetic contractions at 60°·s-1 and 120°·s-1 was significantly higher in dancers and runners than controls. Dancers hopped significantly higher than runners and controls during hopping tasks. Dancers also possessed significantly greater countermovement hop relative peak power, drop hop relative impulse, and drop hop relative peak power than controls. Finally, dancers reached significantly greater velocities during countermovement hops than runners and controls. Our findings suggest dancing and running require or likely enhance plantarflexion strength. Furthermore, dancing appears to require and enhance ankle joint stretch-shortening cycle performance and tendon elongation.

Comparative analysis of the transcriptomes of EDL, psoas, and soleus muscles from mice.

BACKGROUND: Individual skeletal muscles have evolved to perform specific tasks based on their molecular composition. In general, muscle fibers are characterized as either fast-twitch or slow-twitch based on their myosin heavy chain isoform profiles. This approach made sense in the early days of muscle studies when SDS-PAGE was the primary tool for mapping fiber type. However, Next Generation Sequencing tools permit analysis of the entire muscle transcriptome in a single sample, which allows for more precise characterization of differences among fiber types, including distinguishing between different isoforms of specific proteins. We demonstrate the power of this approach by comparing the differential gene expression patterns of extensor digitorum longus (EDL), psoas, and soleus from mice using high throughput RNA sequencing. RESULTS: EDL and psoas are typically classified as fast-twitch muscles based on their myosin expression pattern, while soleus is considered a slow-twitch muscle. The majority of the transcriptomic variability aligns with the fast-twitch and slow-twitch characterization. However, psoas and EDL exhibit unique expression patterns associated with the genes coding for extracellular matrix, myofibril, transcription, translation, striated muscle adaptation, mitochondrion distribution, and metabolism. Furthermore, significant expression differences between psoas and EDL were observed in genes coding for myosin light chain, troponin, tropomyosin isoforms, and several genes encoding the constituents of the Z-disk. CONCLUSIONS: The observations highlight the intricate molecular nature of skeletal muscles and demonstrate the importance of utilizing transcriptomic information as a tool for skeletal muscle characterization.

Calcium-dependent titin-thin filament interactions in muscle: observations and theory.

Gaps in our understanding of muscle mechanics demonstrate that the current model is incomplete. Increasingly, it appears that a role for titin in active muscle contraction might help to fill these gaps. While such a role for titin is increasingly accepted, the underlying molecular mechanisms remain unclear. The goals of this paper are to review recent studies demonstrating Ca2+-dependent interactions between N2A titin and actin in vitro, to explore theoretical predictions of muscle behavior based on this interaction, and to review experimental data related to the predictions. In a recent study, we demonstrated that Ca2+ increases the association constant between N2A titin and F-actin; that Ca2+ increases rupture forces between N2A titin and F-actin; and that Ca2+ and N2A titin reduce sliding velocity of F-actin and reconstituted thin filaments in motility assays. Preliminary data support a role for Ig83, but other Ig domains in the N2A region may also be involved. Two mechanical consequences are inescapable if N2A titin binds to thin filaments in active muscle sarcomeres: (1) the length of titin's freely extensible I-band should decrease upon muscle activation; and (2) binding between N2A titin and thin filaments should increase titin stiffness in active muscle. Experimental observations demonstrate that these properties characterize wild type muscles, but not muscles from mdm mice with a small deletion in N2A titin, including part of Ig83. Given the new in vitro evidence for Ca2+-dependent binding between N2A titin and actin, it is time for skepticism to give way to further investigation.

Effects of a titin mutation on force enhancement and force depression in mouse soleus muscles.

The active isometric force produced by muscles varies with muscle length in accordance with the force-length relationship. Compared with isometric contractions at the same final length, force increases after active lengthening (force enhancement) and decreases after active shortening (force depression). In addition to cross-bridges, titin has been suggested to contribute to force enhancement and depression. Although titin is too compliant in passive muscles to contribute to active tension at short sarcomere lengths on the ascending limb and plateau of the force-length relationship, recent evidence suggests that activation increases titin stiffness. To test the hypothesis that titin plays a role in force enhancement and depression, we investigated isovelocity stretching and shortening in active and passive wild-type and mdm (muscular dystrophy with myositis) soleus muscles. Skeletal muscles from mdm mice have a small deletion in the N2A region of titin and show no increase in titin stiffness during active stretch. We found that: (1) force enhancement and depression were reduced in mdm soleus compared with wild-type muscles relative to passive force after stretch or shortening to the same final length; (2) force enhancement and force depression increased with amplitude of stretch across all activation levels in wild-type muscles; and (3) maximum shortening velocity of wild-type and mdm muscles estimated from isovelocity experiments was similar, although active stress was reduced in mdm compared with wild-type muscles. The results of this study suggest a role for titin in force enhancement and depression, which contribute importantly to muscle force during natural movements.

N2A Titin: Signaling Hub and Mechanical Switch in Skeletal Muscle.

Since its belated discovery, our understanding of the giant protein titin has grown exponentially from its humble beginning as a sarcomeric scaffold to recent recognition of its critical mechanical and signaling functions in active muscle. One uniquely useful model to unravel titin's functions, muscular dystrophy with myositis (mdm), arose spontaneously in mice as a transposon-like LINE repeat insertion that results in a small deletion in the N2A region of titin. This small deletion profoundly affects hypertrophic signaling and muscle mechanics, thereby providing insights into the function of this specific region and the consequences of its dysfunction. The impact of this mutation is profound, affecting diverse aspects of the phenotype including muscle mechanics, developmental hypertrophy, and thermoregulation. In this review, we explore accumulating evidence that points to the N2A region of titin as a dynamic "switch" that is critical for both mechanical and signaling functions in skeletal muscle. Calcium-dependent binding of N2A titin to actin filaments triggers a cascade of changes in titin that affect mechanical properties such as elastic energy storage and return, as well as hypertrophic signaling. The mdm phenotype also points to the existence of as yet unidentified signaling pathways for muscle hypertrophy and thermoregulation, likely involving titin's PEVK region as well as the N2A signalosome.

Optimal length, calcium sensitivity and twitch characteristics of skeletal muscles from mdm mice with a deletion in N2A titin.

During isometric contractions, the optimal length of skeletal muscles increases with decreasing activation. The underlying mechanism for this phenomenon is thought to be linked to length dependence of Ca2+ sensitivity. Muscular dystrophy with myositis (mdm), a recessive titin mutation in mice, was used as a tool to study the role of titin in activation dependence of optimal length and length dependence of Ca2+ sensitivity. We measured the shift in optimal length between tetanic and twitch stimulation in mdm and wild-type muscles, and the length dependence of Ca2+ sensitivity at short and long sarcomere lengths in mdm and wild-type fiber bundles. The results indicate that the mdm mutation leads to a loss of activation dependence of optimal length without the expected change in length dependence of Ca2+ sensitivity, demonstrating that these properties are not linked, as previously suggested. Furthermore, mdm muscles produced maximum tetanic stress during sub-optimal filament overlap at lengths similar to twitch contractions in both genotypes, but the difference explains less than half of the observed reduction in active force of mdm muscles. Mdm muscles also exhibited increased electromechanical delay, contraction and relaxation times, and decreased rate of force development in twitch contractions. We conclude that the small deletion in titin associated with mdm in skeletal muscles alters force production, suggesting an important regulatory role for titin in active force production. The molecular mechanisms for titin's role in regulating muscle force production remain to be elucidated.

Severe thermoregulatory deficiencies in mice with a deletion in the titin gene TTN.

Muscular dystrophy with myositis (mdm) mice carry a deletion in the N2A region of the gene for the muscle protein titin (TTN), shiver at low frequency, fail to maintain body temperatures (Tb) at ambient temperatures (Ta) <34°C, and have reduced body mass and active muscle stiffness in vivo compared with wild-type (WT) siblings. Impaired shivering thermogenesis (ST) could be due to the mutated titin protein causing more compliant muscles. We hypothesized that non-shivering thermogenesis (NST) is impaired. To characterize the response to cold exposure, we measured Tb and metabolic rate (MR) of WT and mdm mice at four nominal temperatures: 20, 24, 29 and 34°C. Subsequently, we stimulated NST with noradrenaline. Manipulation of Ta revealed an interaction between genotype and MR: mdm mice had higher MRs at 29°C and lower MRs at 24°C compared with WT mice. NST capacity was lower in mdm mice than in WT mice. Using MR data from a previous study, we compared MR of mdm mice with MR of Perognathus longimembris, a mouse species of similar body mass. Our results indicated low MR and reduced NST of mdm mice. These were more pronounced than differences between mdm and WT mice owing to body mass effects on MR and capacity for NST. Correcting MR using Q10 showed that mdm mice had lower MRs than size-matched P. longimembris, indicating that mutated N2A titin causes severe thermoregulatory defects at all levels. Direct effects of the titin mutation lead to lower shivering frequency. Indirect effects likely lead to a lower capacity for NST and increased thermal conductance through decreased body size.

The evolution of bite force in horned lizards: the influence of dietary specialization.

Dietary specialization is an important driver of the morphology and performance of the feeding system in many organisms, yet the evolution of phenotypic specialization has only rarely been examined within a species complex. Horned lizards are considered primarily myrmecophagous (ant eating), but variation in diet among the 17 species of horned lizards (Phrynosoma) makes them an ideal group to examine the relationship between dietary specialization and the resultant morphological and functional changes of the feeding system. In this study, we perform a detailed analysis of the jaw adductor musculature and use a biomechanical model validated with in vivo bite force data to examine the evolution of bite force in Phrynosoma. Our model simulations demonstrate that bite force varies predictably with respect to the gape angle and bite position along the tooth row, with maximal bite forces being attained at lower gape angles and at the posterior tooth positions. Maximal bite forces vary considerably among horned lizards, with highly myrmecophagous species exhibiting very low bite forces. In contrast, members of the short-horned lizard clade are able to bite considerably harder than even closely related dietary generalists. This group appears to be built for performing crushing bites and may represent a divergent morphology adapted for eating hard prey items. The evolutionary loss of processing morphology (teeth, jaw and muscle reduction) and bite force in ant specialists may be a response to the lack of prey processing rather than a functional adaptation per se.

The effects of a skeletal muscle titin mutation on walking in mice.

Titin contributes to sarcomere assembly, muscle signaling, and mechanical properties of muscle. The mdm mouse exhibits a small deletion in the titin gene resulting in dystrophic mutants and phenotypically normal heterozygotes. We examined the effects of this mutation on locomotion to assess how, and if, changes to muscle phenotype explain observed locomotor differences. Mutant mice are much smaller in size than their siblings and gait abnormalities may be driven by differences in limb proportions and/or by changes to muscle phenotype caused by the titin mutation. We quantified differences in walking gait among mdm genotypes and also determined whether genotypes vary in limb morphometrics. Mice were filmed walking, and kinematic and morphological variables were measured. Mutant mice had a smaller range of motion at the ankle, shorter stride lengths, and shorter stance duration, but walked at the same relative speeds as the other genotypes. Although phenotypically similar to wildtype mice, heterozygous mice frequently exhibited intermediate gait mechanics. Morphological differences among genotypes in hindlimb proportions were small and do not explain the locomotor differences. We suggest that differences in locomotion among mdm genotypes are due to changes in muscle phenotype caused by the titin mutation.

Effects of a titin mutation on negative work during stretch-shortening cycles in skeletal muscles.

Negative work occurs in muscles during braking movements such as downhill walking or landing after a jump. When performing negative work during stretch-shortening cycles, viscoelastic structures within muscles store energy during stretch, return a fraction of this energy during shortening and dissipate the remaining energy as heat. Because tendons and extracellular matrix are relatively elastic rather than viscoelastic, energy is mainly dissipated by cross bridges and titin. Recent studies demonstrate that titin stiffness increases in active skeletal muscles, suggesting that titin contributions to negative work may have been underestimated in previous studies. The muscular dystrophy with myositis (mdm) mutation in mice results in a deletion in titin that leads to reduced titin stiffness in active muscle, providing an opportunity to investigate the contribution of titin to negative work in stretch-shortening cycles. Using the work loop technique, extensor digitorum longus and soleus muscles from mdm and wild-type (WT) mice were stimulated during the stretch phase of stretch-shortening cycles to investigate negative work. The results demonstrate that, compared with WT muscles, negative work is reduced in muscles from mdm mice. We suggest that changes in the viscoelastic properties of mdm titin reduce energy storage by muscles during stretch and energy dissipation during shortening. Maximum isometric stress is also reduced in muscles from mdm mice, possibly due to impaired transmission of cross-bridge force, impaired cross-bridge function or both. Functionally, the reduction in negative work could lead to increased muscle damage during eccentric contractions that occur during braking movements.

Effects of activation on the elastic properties of intact soleus muscles with a deletion in titin.

Titin has long been known to contribute to muscle passive tension. Recently, it was also demonstrated that titin-based stiffness increases upon Ca2+ activation of wild-type mouse psoas myofibrils stretched beyond overlap of the thick and thin filaments. In addition, this increase in titin-based stiffness was impaired in single psoas myofibrils from mdm mice, characterized by a deletion in the N2A region of the Ttn gene. Here, we investigated the effects of activation on elastic properties of intact soleus muscles from wild-type and mdm mice to determine whether titin contributes to active muscle stiffness. Using load-clamp experiments, we compared the stress-strain relationships of elastic elements in active and passive muscles during unloading, and quantified the change in stiffness upon activation. Results from wild-type muscles show that upon activation, the elastic modulus increases, elastic elements develop force at 15% shorter lengths, and there was a 2.9-fold increase in the slope of the stress-strain relationship. These results are qualitatively and quantitatively similar to results from single wild-type psoas myofibrils. In contrast, mdm soleus showed no effect of activation on the slope or intercept of the stress-strain relationship, which is consistent with impaired titin activation observed in single mdm psoas myofibrils. Therefore, it is likely that titin plays a role in the increase of active muscle stiffness during rapid unloading. These results are consistent with the idea that, in addition to the thin filaments, titin is activated upon Ca2+ influx in skeletal muscle.