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ABSTRACT 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. 

Abstract 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. 

Abstract Navigating complex terrains requires dynamic interactions between the substrate, musculoskeletal, and sensorimotor systems. Current perturbation studies have mostly used visible terrain height perturbations, which do not allow us to distinguish among the neuromechanical contributions of feedforward control, feedback-mediated, and mechanical perturbation responses. Here, we use treadmill-belt speed perturbations to induce a targeted perturbation to foot speed only, and without terrain-induced changes in joint posture and leg loading at stance onset. Based on previous studies suggesting a proximo-distal gradient in neuromechanical control, we hypothesized that distal joints would exhibit larger changes in joint kinematics, compared to proximal joints. Additionally, we expected birds to use feedforward strategies to increase the intrinsic stability of gait. To test these hypotheses, seven adult guinea fowl were video recorded while walking on a motorized treadmill, during both steady and perturbed trials. Perturbations consisted of repeated exposures to a deceleration and acceleration of the treadmill-belt speed. Surprisingly, we found that joint angular trajectories and center of mass fluctuations remain very similar, despite substantial perturbation of foot velocity by the treadmill belt. Hip joint angular trajectories exhibit the largest changes, with the birds adopting a slightly more flexed position across all perturbed strides. Additionally, we observed increased stride duration across all strides, consistent with feedforward changes in the control strategy. The speed perturbations mainly influenced the timing of stance and swing, with the largest kinematic changes in the strides directly following a deceleration. Our findings do not support the general hypothesis of a proximo-distal gradient in joint control, as distal joint kinematics remain largely unchanged. Instead, we find that leg angular trajectory and the timing of stance and swing are most sensitive to this specific perturbation, and leg length actuation remains largely unchanged. Our results are consistent with modular task-level control of leg length and leg angle actuation, with different neuromechanical control and perturbation sensitivity in each actuation mode. Distal joints appear to be sensitive to changes in vertical loading but not foot fore-aft velocity. Future directions should include in vivo studies of muscle activation and force–length dynamics to provide more direct evidence of the sensorimotor control strategies for stability in response to belt-speed perturbations. 

Abstract 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. 

ABSTRACT 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. 

ABSTRACT 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. 

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 Ca 2+ -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, Ca 2+ -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 [Ca 2+ ] or [Ca 2+ ] 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 [Ca 2+ ] plus BDM compared to [Ca 2+ ] alone at a corresponding activation level, suggesting a contribution from Ca 2+ -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 [Ca 2+ ] 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. 

Abstract 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. 

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.