"Avatar", a Modified Ex vivo Work Loop Experiments Using In vivo Strain and Activation.

Movement behaviors are emergent features of dynamic systems that result from muscle force production and work output. The interplay between neural and mechanical systems occurs at all levels of biological organization concurrently, from the tuning of leg muscle properties while running to the dynamics of the limbs interacting with the ground. Understanding the conditions under which animals shift their neural control strategies toward intrinsic muscle mechanics ('preflexes') in the control hierarchy would allow muscle models to predict in vivo muscle force and work more accurately. To understand in vivo muscle mechanics, ex vivo investigation of muscle force and work under dynamically varying strain and loading conditions similar to in vivo locomotion is required. In vivo strain trajectories typically exhibit abrupt changes (i.e., strain and velocity transients) that arise from interactions among neural activation, musculoskeletal kinematics, and loads applied by the environment. The principal goal of our "avatar" technique is to investigate how muscles function during abrupt changes in strain rate and loading when the contribution of intrinsic mechanical properties to muscle force production may be highest. In the "avatar" technique, the traditional work-loop approach is modified using measured in vivo strain trajectories and electromyographic (EMG) signals from animals during dynamic movements to drive ex vivo muscles through multiple stretch-shortening cycles. This approach is similar to the work-loop technique, except that in vivo strain trajectories are scaled appropriately and imposed on ex vivo mouse muscles attached to a servo motor. This technique allows one to: (1) emulate in vivo strain, activation, stride frequency, and work-loop patterns; (2) vary these patterns to match in vivo force responses most accurately; and (3) vary specific features of strain and/or activation in controlled combinations to test mechanistic hypotheses.

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.

The distinctive mechanical and structural signatures of residual force enhancement in myofibers.

In muscle, titin proteins connect myofilaments together and are thought to be critical for contraction, especially during residual force enhancement (RFE) when force is elevated after an active stretch. We investigated titin's function during contraction using small-angle X-ray diffraction to track structural changes before and after 50% titin cleavage and in the RFE-deficient, mdm titin mutant. We report that the RFE state is structurally distinct from pure isometric contractions, with increased thick filament strain and decreased lattice spacing, most likely caused by elevated titin-based forces. Furthermore, no RFE structural state was detected in mdm muscle. We posit that decreased lattice spacing, increased thick filament stiffness, and increased non-crossbridge forces are the major contributors to RFE. We conclude that titin directly contributes to RFE.

Effects of shortening velocity on the stiffness to force ratio during isometric force redevelopment suggest mechanisms of residual force depression.

Although the phenomenon of residual force depression has been known for decades, the mechanisms remain elusive. In the present study, we investigated mechanisms of residual force depression by measuring the stiffness to force ratio during force redevelopment after shortening at different velocities. The results showed that the slope of the relationship between muscle stiffness and force decreased with decreasing shortening velocity, and the y-intercept increased with decreasing shortening velocity. The differing slopes and y-intercepts indicate that the stiffness to force ratio during isometric force redevelopment depends on the active shortening velocity at a given muscle length and activation level. The greater stiffness to force ratio after active shortening can potentially be explained by weakly-bound cross bridges in the new overlap zone. However, weakly-bound cross bridges are insufficient to explain the reduced slope at the slowest shortening velocity because the reduced velocity should increase the proportion of weakly- to strongly-bound cross bridges, thereby increasing the slope. In addition, if actin distortion caused by active shortening recovers during the force redevelopment period, then the resulting slope should be similar to the non-linear slope of force redevelopment over time. Alternatively, we suggest that a tunable elastic element, such as titin, could potentially explain the results.

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.

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.

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.

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.

Isolated Joint Block Progression Training Improves Leaping Performance in Dancers.

The purpose of this study was to investigate the effect of a 12-week ankle-specific block progression training program on saut de chat leaping performance [leap height, peak power (PP), joint kinetics and kinematics], maximal voluntary isometric plantar flexion (MVIP) strength, and Achilles tendon (AT) stiffness. Dancers (training group n = 7, control group n = 7) performed MVIP at plantarflexed (10◦) and neutral ankle positions (0◦) followed by ramping isometric contractions equipped with ultrasound to assess strength and AT stiffness, respectively. Dancers also performed saut de chat leaps surrounded by 3-D motion capture atop force platforms to determine center of mass and joint kinematics and kinetics. The training group then followed a 12-week ankle-focused program including isometric, dynamic constant external resistance, accentuated eccentric loading, and plyometric training modalities, while the control group continued dancing normally. We found that the training group's saut de chat ankle PP (59.8%), braking ankle stiffness (69.6%), center of mass PP (11.4%), and leap height (12.1%) significantly increased following training. We further found that the training group's MVIP significantly increased at 10◦ (17.0%) and 0◦ (12.2%) along with AT stiffness (29.6%), while aesthetic leaping measures were unchanged (peak split angle, mean trunk angle, trunk angle range). Ankle-specific block progression training appears to benefit saut de chat leaping performance, PP output, ankle-joint kinetics, maximal strength, and AT stiffness, while not affecting kinematic aesthetic measures. We speculate that the combined training blocks elicited physiological changes and enhanced neuromuscular synchronization for increased saut de chat leaping performance in this cohort of dancers.

What is an artificial muscle? A comparison of soft actuators to biological muscles.

Interest in emulating the properties of biological muscles that allow for fast adaptability and control in unstructured environments has motivated researchers to develop new soft actuators, often referred to as 'artificial muscles'. The field of soft robotics is evolving rapidly as new soft actuator designs are published every year. In parallel, recent studies have also provided new insights for understanding biological muscles as 'active' materials whose tunable properties allow them to adapt rapidly to external perturbations. This work presents a comparative study of biological muscles and soft actuators, focusing on those properties that make biological muscles highly adaptable systems. In doing so, we briefly review the latest soft actuation technologies, their actuation mechanisms, and advantages and disadvantages from an operational perspective. Next, we review the latest advances in understanding biological muscles. This presents insight into muscle architecture, the actuation mechanism, and modeling, but more importantly, it provides an understanding of the properties that contribute to adaptability and control. Finally, we conduct a comparative study of biological muscles and soft actuators. Here, we present the accomplishments of each soft actuation technology, the remaining challenges, and future directions. Additionally, this comparative study contributes to providing further insight on soft robotic terms, such as biomimetic actuators, artificial muscles, and conceptualizing a higher level of performance actuator named artificial supermuscle. In conclusion, while soft actuators often have performance metrics such as specific power, efficiency, response time, and others similar to those in muscles, significant challenges remain when finding suitable substitutes for biological muscles, in terms of other factors such as control strategies, onboard energy integration, and thermoregulation.

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.

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.

Strength and power capabilities predict weighted parameter ranking of saut de chat leaping performance in dancers.

Limited research exists on the relationship between aesthetic saut de chat performance and muscle-tendon unit (MTU) characteristics of dancers. We developed a weighted parameter ranking (WPR) tool to incorporate aesthetic leaping aspects (i.e., height, peak split angle, average trunk angle and trunk angle range) for correlation with MTU properties. The purpose was to identify the relationship of saut de chat WPR and leap height with maximal plantarflexion strength, medial gastrocnemius (MG) stiffness, Achilles tendon (AT) stiffness and relative peak power (PP). Dancers (n = 18) performed maximal plantarflexion, short-range stretches and isometric ramping contractions on a dynamometer equipped with ultrasound to determine strength, MG stiffness and AT stiffness, respectively. Subjects then performed saut de chat leaps atop force platforms surrounded by motion capture cameras. A principal component analysis (PCA) was performed to compare WPR variable weightings with PCA results and rankings. Moderate-strong relationships were identified among WPR, maximal plantarflexion strength, MG stiffness and PP. Strong-very strong relationships were also identified between leap height and maximal plantarflexion strength, MG stiffness, AT stiffness, peak split angle and PP. A very strong correlation existed between PCA rankings and WPRs. Practitioners may consider developing strength and power capabilities in dancers to improve leaping.

Non-cross Bridge Viscoelastic Elements Contribute to Muscle Force and Work During Stretch-Shortening Cycles: Evidence From Whole Muscles and Permeabilized Fibers.

The sliding filament-swinging cross bridge theory of skeletal muscle contraction provides a reasonable description of muscle properties during isometric contractions at or near maximum isometric force. However, it fails to predict muscle force during dynamic length changes, implying that the model is not complete. Mounting evidence suggests that, along with cross bridges, a Ca2+-sensitive viscoelastic element, likely the titin protein, contributes to muscle force and work. The purpose of this study was to develop a multi-level approach deploying stretch-shortening cycles (SSCs) to test the hypothesis that, along with cross bridges, Ca2+-sensitive viscoelastic elements in sarcomeres contribute to force and work. Using whole soleus muscles from wild type and mdm mice, which carry a small deletion in the N2A region of titin, we measured the activation- and phase-dependence of enhanced force and work during SSCs with and without doublet stimuli. In wild type muscles, a doublet stimulus led to an increase in peak force and work per cycle, with the largest effects occurring for stimulation during the lengthening phase of SSCs. In contrast, mdm muscles showed neither doublet potentiation features, nor phase-dependence of activation. To further distinguish the contributions of cross bridge and non-cross bridge elements, we performed SSCs on permeabilized psoas fiber bundles activated to different levels using either [Ca2+] or [Ca2+] plus the myosin inhibitor 2,3-butanedione monoxime (BDM). Across activation levels ranging from 15 to 100% of maximum isometric force, peak force, and work per cycle were enhanced for fibers in [Ca2+] plus BDM compared to [Ca2+] alone at a corresponding activation level, suggesting a contribution from Ca2+-sensitive, non-cross bridge, viscoelastic elements. Taken together, our results suggest that a tunable viscoelastic element such as titin contributes to: (1) persistence of force at low [Ca2+] in doublet potentiation; (2) phase- and length-dependence of doublet potentiation observed in wild type muscles and the absence of these effects in mdm muscles; and (3) increased peak force and work per cycle in SSCs. We conclude that non-cross bridge viscoelastic elements, likely titin, contribute substantially to muscle force and work, as well as the phase-dependence of these quantities, during dynamic length changes.

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.

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.

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.

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.