Internal fluid pressure influences muscle contractile force.

Fluid fills intracellular, extracellular, and capillary spaces within muscle. During normal physiological activity, intramuscular fluid pressures develop as muscle exerts a portion of its developed force internally. These pressures, typically ranging between 10 and 250 mmHg, are rarely considered in mechanical models of muscle but have the potential to affect performance by influencing force and work produced during contraction. Here, we test a model of muscle structure in which intramuscular pressure directly influences contractile force. Using a pneumatic cuff, we pressurize muscle midcontraction at 260 mmHg and report the effect on isometric force. Pressurization reduced isometric force at short muscle lengths (e.g., -11.87% of P0 at 0.9 L0), increased force at long lengths (e.g., +3.08% of P0 at 1.25 L0), but had no effect at intermediate muscle lengths ∼1.1-1.15 L0 This variable response to pressurization was qualitatively mimicked by simple physical models of muscle morphology that displayed negative, positive, or neutral responses to pressurization depending on the orientation of reinforcing fibers representing extracellular matrix collagen. These findings show that pressurization can have immediate, significant effects on muscle contractile force and suggest that forces transmitted to the extracellular matrix via pressurized fluid may be important, but largely unacknowledged, determinants of muscle performance in vivo.

Passive skeletal muscle can function as an osmotic engine.

Muscles are composite structures. The protein filaments responsible for force production are bundled within fluid-filled cells, and these cells are wrapped in ordered sleeves of fibrous collagen. Recent models suggest that the mechanical interaction between the intracellular fluid and extracellular collagen is essential to force production in passive skeletal muscle, allowing the material stiffness of extracellular collagen to contribute to passive muscle force at physiologically relevant muscle lengths. Such models lead to the prediction, tested here, that expansion of the fluid compartment within muscles should drive forceful muscle shortening, resulting in the production of mechanical work unassociated with contractile activity. We tested this prediction by experimentally increasing the fluid volumes of isolated bullfrog semimembranosus muscles via osmotically hypotonic bathing solutions. Over time, passive muscles bathed in hypotonic solution widened by 16.44 ± 3.66% (mean ± s.d.) as they took on fluid. Concurrently, muscles shortened by 2.13 ± 0.75% along their line of action, displacing a force-regulated servomotor and doing measurable mechanical work. This behaviour contradicts the expectation for an isotropic biological tissue that would lengthen when internally pressurized, suggesting a functional mechanism analogous to that of engineered pneumatic actuators and highlighting the significance of three-dimensional force transmission in skeletal muscle.

The Multi-Scale, Three-Dimensional Nature of Skeletal Muscle Contraction.

Muscle contraction is a three-dimensional process, as anyone who has observed a bulging muscle knows. Recent studies suggest that the three-dimensional nature of muscle contraction influences its mechanical output. Shape changes and radial forces appear to be important across scales of organization. Muscle architectural gearing is an emerging example of this process.

Passive muscle tension increases in proportion to intramuscular fluid volume.

During extended bouts of exercise, muscle can increase in volume by as much as 20% as vascular fluid moves into the tissue. Recent findings suggest that the fluid content of muscle can influence the mechanics of force production; however, the extent to which natural volume fluctuations should be expected to influence muscle mechanics in vivo remains unclear. Here, using osmotic perturbations of bullfrog muscle, we explored the impacts of physiologically relevant volume fluctuations on a fundamental property of muscle: passive force production. We found that passive force and fluid volume were correlated over a 20% increase in muscle volume, with small changes in volume having significant effects on force (e.g. a 5% volume increase results in a >10% passive force increase). A simple physical model of muscle morphology reproduces these effects. These findings suggest that physiologically relevant fluid fluxes could alter passive muscle mechanics in vivo and affect organismal performance.

Incompressible fluid plays a mechanical role in the development of passive muscle tension.

Over short time scales, muscle fibres maintain a nearly constant volume of intracellular fluid. This fluid is essential to normal biochemical function, but its role in determining the mechanical properties of muscle has been considered in only a few theoretical analyses. Here we investigate the mechanical role of fluid in a fundamental property of muscle, its development of passive tension in response to stretch. We test a model of muscle structure in which incompressible fluid directly influences passive tension by constraining the geometry of intramuscular connective tissues. This interaction is demonstrated using a simple physical model of muscle morphology comprising a fluid-filled bladder wrapped by helical fibres. The behaviour of the model is compared with that of isolated bullfrog muscle subjected to an osmotic perturbation of intracellular fluid volume. Increasing muscle volume by 40% resulted in 69% increased passive tension, occurring in a manner consistent with the behaviour of the model. These observations support the notion that the interaction of connective tissues with the muscle fibres they surround influences the mechanical behaviour of whole muscles, and highlight the role of fluid as a mechanical component of muscle.

Architectural gear ratio depends on actuator spacing in a physical model of pennate muscle.

Pennate muscles are defined by the architectural arrangement of their muscle fibers, which run at an angle to the primary axis of muscle shortening. Pennation angles can vary dynamically over the course of individual contractions, influencing the speed and distance of muscle shortening. Despite their relevance to muscle performance, the physical mechanisms that drive dynamic changes in pennation angle remain poorly understood. Muscle fibers bulge radially as they shorten, a consequence of maintaining a constant internal fluid volume, and we hypothesized that radial interactions between tightly packed muscle fibers are essential to dynamic pennation angle changes. To explore this, we built physical models of pennate muscles in which the radial distance between fiber-like actuators could be experimentally altered. Models were built from pennate arrays of McKibben actuators, a type of pneumatic actuator that forcefully shortens and bulges radially when inflated with compressed air. Consistent with past studies of biological muscle and engineered pennate actuators, we found that the magnitude of pennation angle change during contraction varied with load. Importantly, however, we found that pennation angle changes were also strongly influenced by the radial distance between neighboring McKibben actuators. Increasing the radial distance between neighboring actuators reduced pennation angle change during contraction and effectively eliminated variable responses to load. Radial interactions between muscle fibers are rarely considered in theoretical and experimental analyses of pennate muscle; however, these findings suggest that radial interactions between fibers drive pennation angle changes and influence pennate muscle performance. Our results provide insight into the fundamental mechanism underlying dynamic pennation angle changes in biological muscle and highlight design considerations that can inform the development of engineered pennate arrays.

Exploring the Dual Functionality of Plant Pulvini Using a Physical Modeling Approach.

Pulvini are plant motor organs that fulfill two conflicting mechanical roles. At rest, pulvini function as rigid beams that support the cantilevered weight of leafy appendages. During thigmonastic (touch-induced) or nyctinastic ("sleep"-induced) plant movements, however, pulvini function as flexible joints capable of active bending. I hypothesized that the ability to alternate between these roles emerges from the interaction of two structural features of pulvini: anisotropically reinforced parenchyma cells comprising the body of the pulvinus and a longitudinally stiff but flexurally pliant vascular bundle running through the pulvinus core. To investigate how these two components might interact within biological pulvini, I built a set of pulvinus-inspired physical models with varying combinations of these elements present. I compared the abilities of the models to (1) resist imposed bending deformation (i.e., act as rigid beams) and (2) exhibit bending deformation when asymmetrically pressurized (i.e., act as actively deformable joints). Pulvinus models displayed the greatest ability to resist bending deformation when both an anisotropically reinforced parenchyma and a vasculature-like core were present. Disruption of either element reduced hydrostatic fluid pressures developed within the models, resulting in a decreased ability to resist externally applied forces. When differentially pressurized to induce active bending, the degree of bending achieved varied widely between models with and without adequately reinforced parenchyma elements. Bending, however, was not influenced by the presence of a vasculature-like core. These findings suggest that biological pulvini achieve their dual functionality by pairing anisotropically reinforced parenchyma tissues with a longitudinally stiff but flexurally pliant vascular core. Together, these elements compose a hydrostatic skeleton within the pulvinus that strongly resists external deformation when pressurized, but that bends easily when the balance of fluid pressures within it is altered. These results illustrate the emergent nature of pulvinus motor abilities and highlight structural specialization as an important aspect of pulvinus physiology.

Multiscale structural anisotropy steers plant organ actuation.

Leaf movement in vascular plants is executed by joint-like structures called pulvini. Many structural features of pulvini have been described at subcellular, cellular, and tissue scales of organization; however, how the characteristic hierarchical architecture of plant tissue influences pulvinus-mediated actuation remains poorly understood. To investigate the influence of multiscale structure on turgor-driven pulvinus movements, we visualized Mimosa pudica pulvinus morphology and anatomy at multiple hierarchical scales of organization and used osmotic perturbations to experimentally swell pulvini in incremental states of dissection. We observed directional cellulose microfibril reinforcement, oblong, spindle-shaped primary pit fields, and longitudinally slightly compressed cell geometries in the parenchyma of M. pudica. Consistent with these observations, isolated parenchyma tissues displayed highly anisotropic swelling behaviors indicating a high degree of mechanical anisotropy. Swelling behaviors at higher scales of pulvinus organization were also influenced by the presence of the pulvinus epidermis, which displayed oblong epidermal cells oriented transverse to the pulvinus long axis. Our findings indicate that structural specializations spanning multiple hierarchical scales of organization guide hydraulic deformation of pulvini, suggesting that multiscale mechanics are crucial to the translation of cell-level turgor variations into organ-scale pulvinus motion in vivo.

Collagen type VI regulates TGFß bioavailability in skeletal muscle.

Collagen VI-related disorders (COL6-RDs) are a group of rare muscular dystrophies caused by pathogenic variants in collagen VI genes (COL6A1, COL6A2, and COL6A3). Collagen type VI is a heterotrimeric, microfibrillar component of the muscle extracellular matrix (ECM), predominantly secreted by resident fibroadipogenic precursor cells in skeletal muscle. The absence or mislocalizatoion of collagen VI in the ECM underlies the non-cell autonomous dysfunction and dystrophic changes in skeletal muscle with an as of yet elusive direct mechanistic link between the ECM and myofiber dysfunction. Here, we conduct a comprehensive natural history and outcome study in a novel mouse model of COL6-RDs (Col6a2-/- mice) using standardized (Treat-NMD) functional, histological, and physiologic parameter. Notably, we identify a conspicuous dysregulation of the TGFß pathway early in the disease process and propose that the collagen VI deficient matrix is not capable of regulating the dynamic TGFß bioavailability at baseline and also in response to muscle injury. Thus, we propose a new mechanism for pathogenesis of the disease that links the ECM regulation of TGFß with downstream skeletal muscle abnormalities, paving the way for developing and validating therapeutics that target this pathway.

Youth in the study of comparative physiology: insights from demography in the wild.

Of all the properties of individual animals of interest to comparative physiologists, age and stage of development are among the most consequential. In a natural population of any species, the survivorship curve is an important determinant of the relative abundances of ages and stages of development. Demography, thus, has significant implications for the study of comparative physiology. When Edward Deevey published his influential summary of survivorship in animal populations in the wild seven decades ago, he emphasized "serious deficiencies" because survivorship curves for natural populations at the time did not include data on the earliest life stages. Such data have accumulated over intervening years. We survey, for the first time, empirical knowledge of early-age survivorship in populations of most major animal groups in a state of nature. Despite wide variation, it is almost universally true that > 50% of newly born or hatched individuals die before the onset of sexual maturity, even in species commonly assumed to exhibit high early-age survivorship. These demographic facts are important considerations for studies in comparative and environmental physiology whether physiologists (i) aim to elucidate function throughout the life cycle, including both early stages and adults, or (ii) focus on adults (in which case early-age survivorship can potentially affect adult characteristics through selection or epigenesis). We establish that Deevey's Type I curve (which applies to species with relatively limited early mortality) has few or no actual analogs in the real, natural world.

Gastrocnemius Muscle Structural and Functional Changes Associated with Domestication in the Turkey.

Selection for increased muscle mass in domestic turkeys has resulted in muscles twice the size of those found in wild turkeys. This study characterizes muscle structural changes as well as functional differences in muscle performance associated with selection for increased muscle mass. We compared peak isometric force production, whole muscle and individual fiber cross-sectional area (CSA), connective tissue collagen concentration and structure of the lateral gastrocnemius (LG) muscle in wild and adult domestic turkeys. We also explored changes with age between juvenile and adult domestic turkeys. We found that the domestic turkey's LG muscle can produce the same force per cross-sectional area as a wild turkey; however, due to scaling, domestic adults produce less force per unit body mass. Domestic turkey muscle fibers were slightly smaller in CSA (3802 ± 2223 µm2) than those of the wild turkey (4014 ± 1831 µm2, p = 0.013), indicating that the absolutely larger domestic turkey muscles are a result of an increased number of smaller fibers. Collagen concentration in domestic turkey muscle (4.19 ± 1.58 µg hydroxyproline/mg muscle) was significantly lower than in the wild turkeys (6.23 ± 0.63 µg/mg, p = 0.0275), with visible differences in endomysium texture, observed via scanning electron microscopy. Selection for increased muscle mass has altered the structure of the LG muscle; however, scaling likely contributes more to hind limb functional differences observed in the domestic turkey.

Diversity of extracellular matrix morphology in vertebrate skeletal muscle.

Existing data suggest the extracellular matrix (ECM) of vertebrate skeletal muscle consists of several morphologically distinct layers: an endomysium, perimysium, and epimysium surrounding muscle fibers, fascicles, and whole muscles, respectively. These ECM layers are hypothesized to serve important functional roles within muscle, influencing passive mechanics, providing avenues for force transmission, and influencing dynamic shape changes during contraction. The morphology of the skeletal muscle ECM is well described in mammals and birds; however, ECM morphology in other vertebrate groups including amphibians, fish, and reptiles remains largely unexamined. It remains unclear whether a multilayered ECM is a common feature of vertebrate skeletal muscle, and whether functional roles attributed to the ECM should be considered in mechanical analyses of non-mammalian and non-avian muscle. To explore the prevalence of a multilayered ECM, we used a cell maceration and scanning electron microscopy technique to visualize the organization of ECM collagen in muscle from six vertebrates: bullfrogs (Lithobates catesbeianus), turkeys (Meleagris gallopavo), alligators (Alligator mississippiensis), cane toads (Rhinella marina), laboratory mice (Mus musculus), and carp (Cyprinus carpio). All muscles studied contained a collagen-reinforced ECM with multiple morphologically distinct layers. An endomysium surrounding muscle fibers was apparent in all samples. A perimysium surrounding groups of muscle fibers was apparent in all but carp epaxial muscle; a muscle anatomically, functionally, and phylogenetically distinct from the others studied. An epimysium was apparent in all samples taken at the muscle periphery. These findings show that a multilayered ECM is a common feature of vertebrate muscle and suggest that a functionally relevant ECM should be considered in mechanical models of vertebrate muscle generally. It remains unclear whether cross-species variations in ECM architecture are the result of phylogenetic, anatomical, or functional differences, but understanding the influence of such variation on muscle mechanics may prove a fruitful area for future research.

The time course of calf muscle fluid volume during prolonged running.

Muscle fluid is essential for the biochemistry and the biomechanics of muscle contraction. Here, we provide evidence that muscle fluid volumes undergo significant changes during 75 min of moderate intensity (2.7 ± 0.4 m/s) running. Using MRI measurements at baseline and after 2.5, 5, 10, 15, 45 and 75 min, we found that the volumes of calf muscles (quantified through average cross-sectional area) in 18 young recreational runners increase (up to 9% in the gastrocnemii) at the beginning and decrease (below baseline levels) at later stages of running. However, the intensity of changes varied between analyzed muscles. We speculate that these changes are induced by muscle activity and dehydration-related changes in osmotic pressure gradients between intramuscular and extramuscular spaces. These findings highlight the complex nature of muscle fluid shifts during prolonged running exercise.

Release of fascial compartment boundaries reduces muscle force output.

Most limb muscles operate within a compartment defined by fascial layers that enclose a muscle or groups of muscles within a defined space. These compartments are important clinically, because fluid accumulation can cause ischemia and tissue necrosis if untreated. Little is known, however, about how fascial enclosures influence healthy muscle function. One previous study showed that removing a fascial covering reduced the force output of a muscle under maximal stimulation. We hypothesized that such reduction in force output was due to a change in the muscle length following fasciotomy and that a reduced force output could be explained by the length-tension relationship of muscle. Thus we predicted that the maximum force across a range of lengths would be unchanged following fasciotomy. We measured maximal tetanic force output in a wing muscle in wild turkeys both before and after removal of fascia that enclosed the muscle in a compartment. Our hypothesis was not supported. The length-tension curve of this muscle showed that removal of fascia reduced maximum force output to 72 ± 10% of the prefascial release condition. Thus a reduction in muscle force following fasciotomy was not explained by a change in muscle length. The mechanism underlying reduction in force is unclear, but it suggests that the assumption underlying most isolated muscle experiments, i.e., removal of a muscle from its situation in vivo does not influence its maximal mechanical output, may need reexamining. NEW & NOTEWORTHY Most limb muscles are enclosed within compartments bound by robust fascial sheets. The mechanical significance of the close packing of muscle and fascia is largely unexplored. We used an animal model to show that removal of a fascial covering reduces the maximal force developed during contraction. These results raise questions about the use of isolated muscles to estimate muscle performance and suggest that a muscle's mechanical surrounding influences performance by mechanisms that are not understood.