Active muscle stiffness is reduced during rapid unloading in muscles from TtnΔ112-158 mice with a large deletion to PEVK titin.

Evidence suggests that the giant muscle protein titin functions as a tunable spring in active muscle. However, the mechanisms for increasing titin stiffness with activation are not well understood. Previous studies have suggested that during muscle activation, titin binds to actin, which engages the PEVK region of titin, thereby increasing titin stiffness. In this study, we investigated the role of PEVK titin in active muscle stiffness during rapid unloading. We measured elastic recoil of active and passive soleus muscles from TtnΔ112-158 mice characterized by a 75% deletion of PEVK titin and increased passive stiffness. We hypothesized that activated TtnΔ112-158 muscles are stiffer than wild-type muscles as a result of the increased stiffness of PEVK titin. Using a servomotor force lever, we compared the stress-strain relationships of elastic elements in active and passive muscles during rapid unloading and quantified the change in stiffness upon activation. The results show that the elastic modulus of TtnΔ112-158 muscles increased with activation. However, elastic elements developed force at 7% longer lengths and exhibited 50% lower active stiffness in TtnΔ112-158 soleus muscles than in wild-type muscles. Thus, despite having a shorter, stiffer PEVK segment, during rapid unloading, TtnΔ112-158 soleus muscles exhibited reduced active stiffness compared with wild-type soleus muscles. These results are consistent with the idea that PEVK titin contributes to active muscle stiffness; however, the reduction in active stiffness of TtnΔ112-158 muscles suggests that other mechanisms compensate for the increased PEVK stiffness.

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.

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.

Muscle Function from Organisms to Molecules.

Gaps in our understanding of muscle contraction at the molecular level limit the ability to predict in vivo muscle forces in humans and animals during natural movements. Because muscles function as motors, springs, brakes, or struts, it is not surprising that uncertainties remain as to how sarcomeres produce these different behaviors. Current theories fail to explain why a single extra stimulus, added shortly after the onset of a train of stimuli, doubles the rate of force development. When stretch and doublet stimulation are combined in a work loop, muscle force doubles and work increases by 50% per cycle, yet no theory explains why this occurs. Current theories also fail to predict persistent increases in force after stretch and decreases in force after shortening. Early studies suggested that all of the instantaneous elasticity of muscle resides in the cross-bridges. Subsequent cross-bridge models explained the increase in force during active stretch, but required ad hoc assumptions that are now thought to be unreasonable. Recent estimates suggest that cross-bridges account for only ∼12% of the energy stored by muscles during active stretch. The inability of cross-bridges to account for the increase in force that persists after active stretching led to development of the sarcomere inhomogeneity theory. Nearly all predictions of this theory fail, yet the theory persists. In stretch-shortening cycles, muscles with similar activation and contractile properties function as motors or brakes. A change in the phase of activation relative to the phase of length changes can convert a muscle from a motor into a spring or brake. Based on these considerations, it is apparent that the current paradigm of muscle mechanics is incomplete. Recent advances in our understanding of giant muscle proteins, including twitchin and titin, allow us to expand our vision beyond cross-bridges to understand how muscles contribute to the biomechanics and control of movement.

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 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.

Rapid plant evolution in the presence of an introduced species alters community composition.

Because introduced species may strongly interact with native species and thus affect their fitness, it is important to examine how these interactions can cascade to have ecological and evolutionary consequences for whole communities. Here, we examine the interactions among introduced Rocky Mountain elk, Cervus canadensis nelsoni, a common native plant, Solidago velutina, and the diverse plant-associated community of arthropods. While introduced species are recognized as one of the biggest threats to native ecosystems, relatively few studies have investigated an evolutionary mechanism by which introduced species alter native communities. Here, we use a common garden design that addresses and supports two hypotheses. First, native S. velutina has rapidly evolved in the presence of introduced elk. We found that plants originating from sites with introduced elk flowered nearly 3 weeks before plants originating from sites without elk. Second, evolution of S. velutina results in a change to the plant-associated arthropod community. We found that plants originating from sites with introduced elk supported an arthropod community that had ~35 % fewer total individuals and a different species composition. Our results show that the impacts of introduced species can have both ecological and evolutionary consequences for strongly interacting species that subsequently cascade to affect a much larger community. Such evolutionary consequences are likely to be long-term and difficult to remediate.

Phenomenological models of the dynamics of muscle during isotonic shortening.

We investigated the effectiveness of simple, Hill-type, phenomenological models of the force-length-velocity relationship for simulating measured length trajectories during muscle shortening, and, if so, what forms of the model are most useful. Using isotonic shortening data from mouse soleus and toad depressor mandibulae muscles, we showed that Hill-type models can indeed simulate the shortening trajectories with sufficiently good accuracy. However, we found that the standard form of the Hill-type muscle model, called the force-scaling model, is not a satisfactory choice. Instead, the results support the use of less frequently used models, the f-max scaling model and force-scaling with parallel spring, to simulate the shortening dynamics of muscle.

Is titin a 'winding filament'? A new twist on muscle contraction.

Recent studies have demonstrated a role for the elastic protein titin in active muscle, but the mechanisms by which titin plays this role remain to be elucidated. In active muscle, Ca(2+)-binding has been shown to increase titin stiffness, but the observed increase is too small to explain the increased stiffness of parallel elastic elements upon muscle activation. We propose a 'winding filament' mechanism for titin's role in active muscle. First, we hypothesize that Ca(2+)-dependent binding of titin's N2A region to thin filaments increases titin stiffness by preventing low-force straightening of proximal immunoglobulin domains that occurs during passive stretch. This mechanism explains the difference in length dependence of force between skeletal myofibrils and cardiac myocytes. Second, we hypothesize that cross-bridges serve not only as motors that pull thin filaments towards the M-line, but also as rotors that wind titin on the thin filaments, storing elastic potential energy in PEVK during force development and active stretch. Energy stored during force development can be recovered during active shortening. The winding filament hypothesis accounts for force enhancement during stretch and force depression during shortening, and provides testable predictions that will encourage new directions for research on mechanisms of muscle contraction.

What is the role of titin in active muscle?

Several properties of muscle defy explanation solely based on the sliding filament-swinging cross-bridge theory. Indeed, muscle behaves as though there is a dynamic "spring" within the sarcomeres. We propose a new "winding filament" mechanism for how titin acts, in conjunction with the cross-bridges, as a force-dependent spring. The addition of titin into active sarcomeres resolves many puzzling muscle characteristics.

Development of echolocation and communication vocalizations in the big brown bat, Eptesicus fuscus.

Big brown bats form large maternity colonies of up to 200 mothers and their pups. If pups are separated from their mothers, they can locate each other using vocalizations. The goal of this study was to systematically characterize the development of echolocation and communication calls from birth through adulthood to determine whether they develop from a common precursor at the same or different rates, or whether both types are present initially. Three females and their six pups were isolated from our captive breeding colony. We recorded vocal activity from postnatal day 1 to 35, both when the pups were isolated and when they were reunited with their mothers. At birth, pups exclusively emitted isolation calls, with a fundamental frequency range <20 kHz, and duration >30 ms. By the middle of week 1, different types of vocalizations began to emerge. Starting in week 2, pups in the presence of their mothers emitted sounds that resembled adult communication vocalizations, with a lower frequency range and longer durations than isolation calls or echolocation signals. During weeks 2 and 3, these vocalizations were extremely heterogeneous, suggesting that the pups went through a babbling stage before establishing a repertoire of stereotyped adult vocalizations around week 4. By week 4, vocalizations emitted when pups were alone were identical to adult echolocation signals. Echolocation and communication signals both appear to develop from the isolation call, diverging during week 2 and continuing to develop at different rates for several weeks until the adult vocal repertoire is established.

Prey capture in frogs: alternative strategies, biomechanical trade-offs, and hierarchical decision making.

Frogs exhibit flexible repertoires of prey-capture behavior, which depend primarily on visual analysis of prey attributes. We review three examples of how visual cues are used to modulate prey-capture strategies. (1) Dyscophus guineti modulates tongue aiming in response to prey location. These frogs turn only their heads to apprehend prey located at azimuths <40°. At azimuths >40°, the frogs switch from this strategy to one in which both head and tongue are aimed toward prey. (2) Rana pipiens modulates its feeding behavior in response to prey size, using tongue prehension for capturing small prey but switching to jaw prehension to capture large prey. (3) In Cyclorana novaehollandiae, visual processing of prey attributes involves hierarchical decision making. These frogs first assess prey size. For large prey, they ignore velocity but not shape. For small prey, they ignore shape but not velocity. Alternative prey-capture strategies are associated with biomechanical trade-offs that result from the interaction between the feeding apparatus and varying attributes of prey. Alternative strategies likely exist because biomechanical constraints prevent any one strategy from being effective over a range of prey attributes. Taken together, these studies emphasize the requirement that predators must somehow tune prey-capture kinematics simultaneously to multiple attributes of prey. In frogs, the choice among alternative prey-capture strategies involves a hierarchical decision-making process. Hierarchical decision making is expected to be widespread among animals. However, no previous studies were found except for humans, who frequently use this type of approach to make complex decisions.

Prey location, biomechanical constraints, and motor program choice during prey capture in the tomato frog, Dyscophus guineti.

This study investigated how visual information about prey location and biomechanical constraints of the feeding apparatus influence the feeding behavior of the tomato frog, Dyscophus guineti. When feeding on prey at small azimuths (less than +/- 40 degrees), frogs aimed their heads toward the prey but did not aim their tongues relative to their heads. Frogs projected their tongues rapidly by transferring momentum from the lower jaw to the tongue. Storage and recovery of elastic energy by the mouth opening muscles amplified the velocities of mouth opening and tongue projection. This behavior can only occur when the lower jaw and tongue are aligned (i.e., within the range of motion of the neck). When feeding on prey at large azimuths (greater than +/- 40 degrees), frogs aimed both the head and tongue toward the prey and used a muscular hydrostatic mechanism to project the tongue. Hydrostatic elongation allows for frogs to capture prey at greater azimuthal locations. Because the tongue moves independently of the lower jaw, frogs can no longer take advantage of momentum transfer to amplify the speed of tongue projection. To feed on prey at different azimuthal locations, tomato frogs switch between alternative strategies to circumvent these biomechanical constraints.

Elastic properties of active muscle--on the rebound?

During active lengthening and shortening, muscles exhibit a variety of time-dependent spring properties, including load-dependent and nonlinear stiffness. These properties can be explained as interactions between a spring element and cycling cross bridges within muscle sarcomeres. Several lines of evidence suggest a role for the giant protein titin in active muscle, but specific mechanisms remain to be elucidated.

Storage and recovery of elastic potential energy powers ballistic prey capture in toads.

Ballistic tongue projection in toads is a remarkably fast and powerful movement. The goals of this study were to: (1) quantify in vivo power output and activity of the depressor mandibulae muscles that are responsible for ballistic mouth opening, which powers tongue projection; (2) quantify the elastic properties of the depressor mandibulae muscles and their series connective tissues using in situ muscle stimulation and force-lever studies; and (3) develop and test an elastic recoil model, based on the observed elastic properties of the depressor mandibulae muscles and series connective tissues, that accounts for displacement, velocity, acceleration and power output during ballistic mouth opening in toads. The results demonstrate that the depressor mandibulae muscles of toads are active for up to 250 ms prior to mouth opening. During this time, strains of up to 21.4% muscle resting length (ML) develop in the muscles and series connective tissues. At maximum isometric force, series connective tissues develop strains up to 14% ML, and the muscle itself develops strains up to 17.5% ML. When the mouth opens rapidly, the peak instantaneous power output of the depressor mandibulae muscles and series connective tissues can reach 9600 W kg(-1). The results suggest that: (1) elastic recoil of muscle itself can contribute significantly to the power of ballistic movements; (2) strain in series elastic elements of the depressor mandibulae muscle is too large to be borne entirely by the cross bridges and the actin-myosin filament lattice; and (3) central nervous control of ballistic tongue projection in toads likely requires the specification of relatively few parameters.

Contribution of eye retraction to swallowing performance in the northern leopard frog, Rana pipiens.

Most anurans retract and close their eyes repeatedly during swallowing. Eye retraction may aid swallowing by helping to push food back toward the esophagus, but this hypothesis has never been tested. We used behavioral observations, cineradiography, electromyography and nerve transection experiments to evaluate the contribution of eye retraction to swallowing in the northern leopard frog, Rana pipiens. Behavioral observations of frogs feeding on 1.5 cm long crickets reveal a high degree of variability in eye retraction and swallowing. Eye retraction can occur bilaterally or unilaterally, and both swallowing movements and eye retraction can occur separately as well as together. During swallowing, cineradiography shows that the eyes and associated musculature retract well into the oropharynx and appear to make contact with the prey item. This contact appears to help push the prey toward the esophagus, and it may also serve to anchor the prey for tongue-based transport. Electromyographic recordings confirm strong activity in the retractor bulbi muscles during eye retraction. After bilateral denervation of the retractor bulbi, frogs maintain the ability to swallow but show a 74% increase in the number of swallows required per cricket (from a mean of 2.3 swallows to a mean of 4.0 swallows per cricket). Our results indicate that, in Rana pipiens feeding on medium-sized crickets, eye retraction is an accessory swallowing mechanism that assists the primary tongue-based swallowing mechanism.

Mechanism of tongue protraction in microhylid frogs.

High-speed videography and muscle denervation experiments were used to elucidate the mechanism of tongue protraction in the microhylid frog Phrynomantis bifasciatus. Unlike most frogs, Phrynomantis has the ability to protract the tongue through a lateral arc of over 200 degrees in the frontal plane. Thus, the tongue can be aimed side to side, independently of head and jaw movements. Denervation experiments demonstrate that the m. genioglossus complex controls lateral tongue aiming with a hydrostatic mechanism. After unilateral denervation of the m. genioglossus complex, the tongue can only be protracted towards the denervated (inactive) side and the range through which the tongue can be aimed is reduced by 75%. Histological sections of the tongue reveal a compartment of perpendicularly arranged muscle fibers, the m. genioglossus dorsoventralis. This compartment, in conjunction with the surrounding connective tissue, generates hydrostatic pressure that powers tongue movements in Phrynomantis. A survey of aiming abilities in 17 additional species of microhylid frogs, representing a total of 12 genera and six subfamilies, indicates that hydrostatic tongues are found throughout this family. Among frogs, this mechanism of tongue protraction was previously known only in Hemisus and may represent a synapomorphy of Hemisus and Microhylidae.