Bridging the muscle genome to phenome across multiple biological scales.

Muscle is highly hierarchically organized, with functions shaped by genetically controlled expression of protein ensembles with different isoform profiles at the sarcomere scale. However, it remains unclear how isoform profiles shape whole-muscle performance. We compared two mouse hindlimb muscles, the slow, relatively parallel-fibered soleus and the faster, more pennate-fibered tibialis anterior (TA), across scales: from gene regulation, isoform expression and translation speed, to force-length-velocity-power for intact muscles. Expression of myosin heavy-chain (MHC) isoforms directly corresponded with contraction velocity. The fast-twitch TA with fast MHC isoforms had faster unloaded velocities (actin sliding velocity, Vactin; peak fiber velocity, Vmax) than the slow-twitch soleus. For the soleus, Vactin was biased towards Vactin for purely slow MHC I, despite this muscle's even fast and slow MHC isoform composition. Our multi-scale results clearly identified a consistent and significant dampening in fiber shortening velocities for both muscles, underscoring an indirect correlation between Vactin and fiber Vmax that may be influenced by differences in fiber architecture, along with internal loading due to both passive and active effects. These influences correlate with the increased peak force and power in the slightly more pennate TA, leading to a broader length range of near-optimal force production. Conversely, a greater force-velocity curvature in the near-parallel fibered soleus highlights the fine-tuning by molecular-scale influences including myosin heavy and light chain expression along with whole-muscle characteristics. Our results demonstrate that the individual gene, protein and whole-fiber characteristics do not directly reflect overall muscle performance but that intricate fine-tuning across scales shapes specialized muscle function.

Elastic recoil action amplifies jaw closing speed in an aquatic feeding salamander.

Tendon springs often influence locomotion by amplifying the speed and power of limb joint rotation. However, less is known about elastic recoil action in feeding systems, particularly for small aquatic animals. Here, we ask if elastic recoil amplifies the speed of gape closing during aquatic food processing in the Axolotl (Ambystoma mexicanum). We measure activation of the adductor mandibulae externus via electromyography and strain of the jaw adductor muscle-tendon unit (MTU), and gape kinematics via fluoromicrometry. The muscle is pre-activated coincident with gape opening, which causes MTU stretch. Activation lasts significantly shorter for fish than cricket processing, and muscle shortening during MTU lengthening yields significantly greater elastic strain for cricket processing. The speed of MTU shortening, which dictates the speed of gape closing is 2.5-4.4 times greater than the speed of the initial shortening of the muscle fascicles for fish and cricket gape cycles, respectively. These data demonstrate a clear role for elastic recoil, which may be unexpected for a MTU in a feeding system of a small, aquatic animal. Amplification of jaw-closing speed resulting from elastic recoil likely confers ecological advantages in reducing prey escape risks during food processing in a dense and viscous fluid environment.

Ontogenetic plasticity in cranial morphology is associated with a change in the food processing behavior in Alpine newts.

BACKGROUND: The feeding apparatus of salamanders consists mainly of the cranium, mandible, teeth, hyobranchial apparatus and the muscles of the cranial region. The morphology of the feeding apparatus in turn determines the boundary conditions for possible food processing (i.e., intraoral mechanical reduction) mechanisms. However, the morphology of the feeding apparatus changes substantially during metamorphosis, prompting the hypothesis that larvae might use a different food processing mechanism than post-metamorphic adults. Salamandrid newts with facultative metamorphosis are suitable for testing this hypothesis as adults with divergent feeding apparatus morphologies often coexist in the same population, share similar body sizes, and feed on overlapping prey spectra. METHODS: We use high-speed videography to quantify the in vivo movements of key anatomical elements during food processing in paedomorphic and metamorphic Alpine newts (Ichthyosaura alpestris). Additionally, we use micro-computed tomography (µCT) to analyze morphological differences in the feeding apparatus of paedomorphic and metamorphic Alpine newts and sort them into late-larval, mid-metamorphic and post-metamorphic morphotypes. RESULTS: Late-larval, mid-metamorphic and post-metamorphic individuals exhibited clear morphological differences in their feeding apparatus. Regardless of the paedomorphic state being externally evident, paedomorphic specimens can conceal different morphotypes (i.e., late-larval and mid-metamorphic morphotypes). Though feeding on the same prey under the same (aquatic) condition, food processing kinematics differed between late-larval, mid-metamorphic and post-metamorphic morphotypes. CONCLUSIONS: The food processing mechanism in the Alpine newt changes along with morphology of the feeding apparatus during ontogeny, from a mandible-based to a tongue-based processing mechanism as the changing morphology of the mandible prevents chewing and the tongue allows enhanced protraction. These results could indicate that early tetrapods, in analogy to salamanders, may have developed new feeding mechanisms in their aquatic environment and that these functional innovations may have later paved the way for terrestrial feeding mechanisms.

A salamander that chews using complex, three-dimensional mandible movements.

Most non-mammal tetrapods have a hinge-like jaw operation restricted to vertical opening and closing movements. Many mammal jaw joints, by contrast, operate in more complex, three-dimensional (3D) ways, involving not only vertical but also propalinal (rostro-caudal) and transverse (lateral) movements. Data on intraoral food processing in lissamphibians and sauropsids has prompted a generally accepted view that these groups mostly swallow food unreduced, and that in those cases where lissamphibians and sauropsids chew, they mostly use simple vertical jaw movements for food processing. The exception to this generally accepted view is the occurrence of some propalinal chewing in sauropsids. We combined 3D kinematics and morphological analyses from biplanar high-speed video fluoroscopy and micro-computed tomography to determine how the paedomorphic salamander Siren intermedia treats captured food. We discovered not only that S. intermedia uses intraoral food processing but also that the elaborated morphology of its jaw joint facilitates mandibular motions in all three planes, resulting in complex 3D chewing. Thus, our data challenge the commonly held view that complex 3D chewing movements are exclusive to mammals, by suggesting that such mechanisms might have evolved early in the tetrapod evolution.

Flexibility of intraoral food processing in the salamandrid newt Triturus carnifex: effects of environment and prey type.

Intraoral food processing mechanisms are known for all major vertebrate groups, but the form and function of systems used to crush, grind or puncture food items can differ substantially between and within groups. Most vertebrates display flexible mechanisms of intraoral food processing with respect to different environmental conditions or food types. It has recently been shown that newts use cyclical loop-motions of the tongue to rasp prey against the palatal dentition. However, it remains unknown whether newts can adjust their food processing behavior in response to different food types or environmental conditions. Newts are interesting models for studying the functional adaptation to different conditions because of their unique and flexible lifestyle: they seasonally change between aquatic and terrestrial habitats, adapt their prey-capture mode to the respective environment, and consume diverse food types with different mechanical properties. Using X-ray high-speed recordings, anatomical investigations, behavioral analyses and mechanical property measurements, we tested the effects of the medium in which feeding occurs (water/air) and the food type (maggot, earthworm, cricket) on the processing behavior in Triturus carnifex We discovered that food processing, by contrast to prey capture, differed only slightly between aquatic and terrestrial habitats. However, newts adjusted the number of processing cycles to different prey types: while maggots were processed extensively, earthworm pieces were barely processed at all. We conclude that, in addition to food mechanical properties, sensory feedback such as smell and taste appear to induce flexible processing responses, while the medium in which feeding occurs appears to have less of an effect.

Added mass in rat plantaris muscle causes a reduction in mechanical work.

Most of what we know about whole muscle behaviour comes from experiments on single fibres or small muscles that are scaled up in size without considering the effects of the additional muscle mass. Previous modelling studies have shown that tissue inertia acts to slow the rate of force development and maximum velocity of muscle during shortening contractions and decreases the work and power per cycle during cyclic contractions; however, these results have not yet been confirmed by experiments on living tissue. Therefore, in this study we conducted in situ work-loop experiments on rat plantaris muscle to determine the effects of increasing the mass of muscle on mechanical work during cyclic contractions. We additionally simulated these experimental contractions using a mass-enhanced Hill-type model to validate our previous modelling work. We found that greater added mass resulted in lower mechanical work per cycle relative to the unloaded trials in which no mass was added to the muscle (P=0.041 for both 85 and 123% increases in muscle mass). We additionally found that greater strain resulted in lower work per cycle relative to unloaded trials at the same strain to control for length change and velocity effects on the work output, possibly due to greater accelerations of the muscle mass at higher strains. These results confirm that tissue mass reduces muscle mechanical work at larger muscle sizes, and that this effect is likely amplified for lower activations.

Post-activation muscle potentiation and its relevance to cyclical behaviours.

Muscle can experience post-activation potentiation (PAP), a temporary increase in force and rate of force development, when contractions are closely timed; therefore, cyclical behaviours are likely affected by PAP, as succeeding contraction cycles can lead to potentiation over several subsequent cycles. Here, we examined PAP during in situ cyclical contractions of the mallard lateral gastrocnemius (LG). Surface swimming, a cyclical behaviour, was mimicked with work-loops using in vivo LG length change and stimulation parameters. Tests were performed at mallards' preferred cycle frequency as well as at lower and higher frequencies. Like muscles from mammals, anurans and arthropods, the mallard LG exhibited PAP with increases in peak force, average force rate and net work. Staircase potentiation occurred over two or more work-loop cycles, resulting in gradual increases in PAP. The number of cycles needed to reach maximum work varied with cycle frequency, requiring more cycles at higher cycle frequencies. PAP occurred under in vivo-like stimulation parameters, suggesting a potentially important role of PAP in animal locomotion, especially in cyclical behaviours.

Evidence of a tunable biological spring: elastic energy storage in aponeuroses varies with transverse strain in vivo.

Tendinous structures are generally thought of as biological springs that operate with a fixed stiffness, yet recent observations on the mechanical behaviour of aponeuroses (broad, sheet-like tendons) have challenged this general assumption. During in situ contractions, aponeuroses undergo changes in both length and width and changes in aponeuroses width can drive changes in longitudinal stiffness. Here, we explore if changes in aponeuroses width can modulate elastic energy (EE) storage in the longitudinal direction. We tested this idea in vivo by quantifying muscle and aponeuroses mechanical behaviour in the turkey lateral gastrocnemius during landing and jumping, activities that require rapid rates of energy dissipation and generation, respectively. We discovered that when aponeurosis width increased (as opposed to decreased), apparent longitudinal stiffness was 34% higher and the capacity of aponeuroses to store EE when stretched in the longitudinal direction was 15% lower. These data reveal that biaxial loading of aponeuroses allows for variation in tendon stiffness and energy storage for different locomotor behaviours.

Chewing or not? Intraoral food processing in a salamandrid newt.

Food processing refers to any form of mechanical breakdown of food prior to swallowing. Variations of this behaviour are found within all major gnathostome groups. Chewing is by far the most commonly used intraoral processing mechanism and involves rhythmic mandibular jaw and hyobranchial (tongue) movements. Chewing occurs in chondrichthyans (sharks and rays), actinopterygians (ray-finned fishes), dipnoi (lungfishes) as well as amniotes and involves similarities in the patterns of muscle activity and movement of the feeding apparatus. It has been suggested that amniote chewing, which involves the interaction of movements of the mandibular jaw and the muscular tongue, has evolved as part of the tetrapod land invasion. However, little is known about food-processing mechanisms in lissamphibians, which might have retained many ancestral tetrapod features. Here, we identified a processing mechanism in the salamandrid newt, Triturus carnifex, which after prey capture displays cyclic head bobbing in concert with rhythmic jaw and tongue movements. We used high-speed fluoroscopy, anatomical reconstructions and analyses of stomach contents to show that newts, although not using their mandibular jaws, deploy a derived processing mechanism where prey items are rasped rhythmically against the dentition on the mouth roof, driven by a loop motion of the tongue. We then compared patterns and coordination of jaw and tongue movements across gnathostomes to conclude that food processing in this newt species shares traits with processing mechanisms in fish as well as amniotes. This discovery casts salamanders as promising models for reconstructing the evolution of intraoral processing mechanisms at the fish-tetrapod split.

Experimental evidence that physical activity affects the multivariate associations among muscle attachments (entheses).

The morphology of entheses (muscle/tendon attachment sites) on bones is routinely used in paleontological and bioarcheological studies to infer the physical activity patterns of ancient vertebrate species including hominins. However, such inferences have often been disputed owing to limitations of the quantitative methods commonly employed and a lack of experimental evidence demonstrating direct effects of physical activity on entheseal morphology. Recently, we introduced a new and improved method of quantifying and analyzing entheseal morphology that involves repeatable three-dimensional measurements combined with multivariate statistics focused on associations among multiple entheses. Here, to assess the validity of our method for investigating variation in entheseal morphology related to physical activity patterns, we analyzed femora of growing turkeys that were experimentally exercised for 10 weeks on either an inclined or declined treadmill or served as controls (N=15 individuals, 5 per group). Our multivariate approach identified certain patterns involving three different entheses (associated with the gluteus primus, medial gastrocnemius, vastus medialis and adductor magnus muscles) that clearly differentiated controls from runners. Importantly, these differences were not observable when comparing groups within each of the three entheseal structures separately. Body mass was not correlated with the resulting multivariate patterns. These results provide the first experimental evidence that variation in physical activity patterns has a direct influence on entheseal morphology. Moreover, our findings highlight the promise of our newly developed quantitative methods for analyzing the morphology of entheses to reconstruct the behavior of extinct vertebrate species based on their skeletal remains.