Phalloidin suppresses force in nebulin-rich lamprey cardiac muscle

We assessed the effect of phalloidin, known to detach nebulin from actin in skeletal myofibrils, on the isometric force of skinned lamprey cardiac muscle, which has nebulin in amounts comparable to that in skeletal muscle. In contrast to mammalian cardiac muscle, which contains much less nebulin and reacts to phalloidin only by an increase in force, the lamprey cardiac muscle responds to phalloidin by a pronounced (～ 50%) reduction in isometric force, thereby resembling the behavior of skeletal muscle. These results support our hypothesis that nebulin detachment from actin underlies phalloidin-induced force loss and suggest a role of actin-nebulin interaction in contractile function.     

A comparison of the mechanical behavior of the cat soleus muscle with a distribution-moment model

A state-variable model for skeletal muscle, termed the "Distribution-Moment Model," is derived from A. F. Huxley's 1957 model of molecular contraction dynamics. The state variables are the muscle stretch and the three lowest-order moments of the bond-distribution function (which represent, respectively, the contractile tissue stiffness, the muscle force, and the elastic energy stored in the contractile tissue). The rate equations of the model are solved under various conditions, and compared to experimental results for the cat soleus muscle subjected to constant stimulation. The model predicts several observed effects, including yielding of the muscle force in constant velocity stretches, different "force-velocity relations" in isotonic and isovelocity experiments, and a decrease of peak force below the isometric level in small-amplitude sinusoidal stretches. Chemical energy and heat rates predicted by the model are also presented.     

Model representation of the nonlinear step response in cardiac muscle

Motivated by the need for an analytical tool that can be used routinely to analyze data collected from isolated, detergent-skinned cardiac muscle fibers, we developed a mathematical model for representing the force response to step changes in muscle length (i.e., quick stretch and release). Our proposed model is reasonably simple, consisting of only five parameters representing: (1) the rate constant by which length change–induced distortion of elastic elements is dissipated; (2) the stiffness of the muscle fiber; (3) the amplitude of length-mediated recruitment of stiffness elements; (4) the rate constant by which this length-mediated recruitment takes place; and (5) the magnitude of the nonlinear interaction term by which distortion of elastic elements affects the number of recruited stiffness elements. Fitting this model to a family of force recordings representing responses to eight amplitudes of step length change (±2.0% baseline muscle length in 0.5% increments) enabled four things: (1) reproduction of all the identifiable features seen in a family of force responses to both positive and negative length changes; (2) close fitting of all records from the whole family of these responses with very little residual error; (3) estimation of all five model parameters with a great degree of certainty; and (4) importantly, ready discrimination between cardiac muscle fibers with different contractile regulatory proteins but showing only subtly different contractile function. We recommend this mathematical model as an analytic tool for routine use in studies of cardiac muscle fiber contractile function. Such model-based analysis gives novel insight to the contractile behavior of cardiac muscle fibers, and it is useful for characterizing the mechanistic effects that alterations of cardiac contractile proteins have on cardiac contractile function.     

Leucine elicits myotube hypertrophy and enhances maximal contractile force in tissue engineered skeletal muscle in vitro

The amino acid leucine is thought to be important for skeletal muscle growth by virtue of its ability to acutely activate mTORC1 and enhance muscle protein synthesis, yet little data exist regarding its impact on skeletal muscle size and its ability to produce force. We utilized a tissue engineering approach in order to test whether supplementing culture medium with leucine could enhance mTORC1 signaling, myotube growth, and muscle function. Phosphorylation of the mTORC1 target proteins 4EBP‐1 and rpS6 and myotube hypertrophy appeared to occur in a dose dependent manner, with 5 and 20 mM of leucine inducing similar effects, which were greater than those seen with 1 mM. Maximal contractile force was also elevated with leucine supplementation; however, although this did not appear to be enhanced with increasing leucine doses, this effect was completely ablated by co‐incubation with the mTOR inhibitor rapamycin, showing that the augmented force production in the presence of leucine was mTOR sensitive. Finally, by using electrical stimulation to induce chronic (24 hr) contraction of engineered skeletal muscle constructs, we were able to show that the effects of leucine and muscle contraction are additive, since the two stimuli had cumulative effects on maximal contractile force production. These results extend our current knowledge of the efficacy of leucine as an anabolic nutritional aid showing for the first time that leucine supplementation may augment skeletal muscle functional capacity, and furthermore validates the use of engineered skeletal muscle for highly‐controlled investigations into nutritional regulation of muscle physiology.     

Phalloidin suppresses the force in nebulin-rich lamprey cardiac muscle

The effect of phalloidin, an agent detaching nebulin from actin in skeletal muscle, on the isometric force in lamprey skinned cardiac muscle, which has nebulin in amounts comparable to that in skeletal muscle, has been studied. We found that, unlike mammalian cardiac muscle expressing nebulin less abundantly and responding to phalloidin by a force increase, lamprey cardiac muscle responds to phalloidin by a force decrease (approximately 50% decrease), thereby resembling the response of skeletal muscle. These results support our hypothesis that nebulin detachment from actin underlies phalloidin-induced force loss and suggest a role of actin-nebulin interaction in contractile function.     

Genetic Models in Applied Physiology. Merosin deficiency leads to alterations in passive and active skeletal muscle mechanics.

The role of extracellular elements on the mechanical properties of skeletal muscles is unknown. Merosin is an essential extracellular matrix protein that forms a mechanical junction between the sarcolemma and collagen. Therefore, it is possible that merosin plays a role in force transmission between muscle fibers and collagen. We hypothesized that deficiency in merosin may alter passive muscle stiffness, viscoelastic properties, and contractile muscle force in skeletal muscles. We used the dy/dy mouse, a merosin-deficient mouse model, to examine changes in passive and active muscle mechanics. After mice were anesthetized and the diaphragm or the biceps femoris hindlimb muscle was excised, passive length-tension relationships, stress-relaxation curves, or isometric contractile properties were determined with an in vitro biaxial mechanical testing apparatus. Compared with controls, extensibility was smaller in the muscle fiber direction and the transverse fiber direction of the mutant mice. The relaxed elastic modulus was smaller in merosin-deficient diaphragms compared with controls. Interestingly, maximal muscle tetanic stress was depressed in muscles from the mutant mice during uniaxial loading but not during biaxial loading. However, presence of transverse passive stretch increases maximal contractile stress in both the mutant and normal mice. Our data suggest that merosin contributes to muscle passive stiffness, viscoelasticity, and contractility and that the mechanism by which force is transmitted between adjacent myofibers via merosin possibly in shear.     

Micromechanical function of myofibrils isolated from skeletal and cardiac muscles of the zebrafish

The zebrafish is a potentially important and cost-effective model for studies of development, motility, regeneration, and inherited human diseases. The object of our work was to show whether myofibrils isolated from zebrafish striated muscle represent a valid subcellular contractile model. These organelles, which determine contractile function in muscle, were used in a fast kinetic mechanical technique based on an atomic force probe and video microscopy. Mechanical variables measured included rate constants of force development (k(ACT)) after Ca(2+) activation and of force decay (τ(REL)(-1)) during relaxation upon Ca(2+) removal, isometric force at maximal (F(max)) or partial Ca(2+) activations, and force response to an external stretch applied to the relaxed myofibril (F(pass)). Myotomal myofibrils from larvae developed greater active and passive forces, and contracted and relaxed faster than skeletal myofibrils from adult zebrafish, indicating developmental changes in the contractile organelles of the myotomal muscles. Compared with murine cardiac myofibrils, measurements of adult zebrafish ventricular myofibrils show that k(ACT), F(max), Ca(2+) sensitivity of the force, and F(pass) were comparable and τ(REL)(-1) was smaller. These results suggest that cardiac myofibrils from zebrafish, like those from mice, are suitable contractile models to study cardiac function at the sarcomeric level. The results prove the practicability and usefulness of mechanical and kinetic investigations on myofibrils isolated from larval and adult zebrafish muscles. This novel approach for investigating myotomal and myocardial function in zebrafish at the subcellular level, combined with the powerful genetic manipulations that are possible in the zebrafish, will allow the investigation of the functional primary consequences of human disease-related mutations in sarcomeric proteins in the zebrafish model.     

Resistance to radial expansion limits muscle strain and work

The collagenous extracellular matrix (ECM) of skeletal muscle functions to transmit force, protect sensitive structures, and generate passive tension to resist stretch. The mechanical properties of the ECM change with age, atrophy, and neuromuscular pathologies, resulting in an increase in the relative amount of collagen and an increase in stiffness. Although numerous studies have focused on the effect of muscle fibrosis on passive muscle stiffness, few have examined how these structural changes may compromise contractile performance. Here we combine a mathematical model and experimental manipulations to examine how changes in the mechanical properties of the ECM constrain the ability of muscle fibers and fascicles to radially expand and how such a constraint may limit active muscle shortening. We model the mechanical interaction between a contracting muscle and the ECM using a constant volume, pressurized, fiber-wound cylinder. Our model shows that as the proportion of a muscle cross section made up of ECM increases, the muscle’s ability to expand radially is compromised, which in turn restricts muscle shortening. In our experiments, we use a physical constraint placed around the muscle to restrict radial expansion during a contraction. Our experimental results are consistent with model predictions and show that muscles restricted from radial expansion undergo less shortening and generate less mechanical work under identical loads and stimulation conditions. This work highlights the intimate mechanical interaction between contractile and connective tissue structures within skeletal muscle and shows how a deviation from a healthy, well-tuned relationship can compromise performance.     

Cooperative attachment of cross bridges predicts regulation of smooth muscle force by myosin phosphorylation

Serine 19 phosphorylation of the myosin regulatory light chain (MRLC) appears to be the primary determinant of smooth muscle force development. The relationship between MRLC phosphorylation and force is nonlinear, showing that phosphorylation is not a simple switch regulating the number of cycling cross bridges. We reexamined the MRLC phosphorylation-force relationship in slow, tonic swine carotid media; fast, phasic rabbit urinary bladder detrusor; and very fast, tonic rat anococcygeus. We found a sigmoidal dependence of force on MRLC phosphorylation in all three tissues with a threshold for force development of approximately 0.15 mol P(i)/mol MRLC. This behavior suggests that force is regulated in a highly cooperative manner. We then determined whether a model that employs both the latch-bridge hypothesis and cooperative activation could reproduce the relationship between Ser(19)-MRLC phosphorylation and force without the need for a second regulatory system. We based this model on skeletal muscle in which attached cross bridges cooperatively activate thin filaments to facilitate cross-bridge attachment. We found that such a model describes both the steady-state and time-course relationship between Ser(19)-MRLC phosphorylation and force. The model required both cooperative activation and latch-bridge formation to predict force. The best fit of the model occurred when binding of a cross bridge cooperatively activated seven myosin binding sites on the thin filament. This result suggests cooperative mechanisms analogous to skeletal muscle that will require testing.     

Three-dimensional finite element modeling of skeletal muscle using a two-domain approach: linked fiber-matrix mesh model

In previous applications of the finite element method in modeling mechanical behavior of skeletal muscle, the passive and active properties of muscle tissue were lumped in one finite element. Although this approach yields increased understanding of effects of force transmission, it does not support an assessment of the interaction between the intracellular structures and extracellular matrix. In the present study, skeletal muscle is considered in two domains: (1) the intracellular domain and (2) extracellular matrix domain. The two domains are represented by two separate meshes that are linked elastically to account for the trans-sarcolemmal attachments of the muscle fibers' cytoskeleton and extracellular matrix. With this approach a finite element skeletal muscle model is developed, which allows force transmission between these domains with the possibility of investigating their interaction as well as the role of the trans-sarcolemmal systems. The model is applied to show the significance of myofascial force transmission by investigating possible mechanical consequences due to any missing link within the trans-sarcolemmal connections such as found in muscular dystrophies. This is realized by making the links between the two meshes highly compliant at selected intramuscular locations. The results indicate the role of extracellular matrix for a muscle in sustaining its physiological condition. It is shown that if there is an inadequate linking to the extracellular matrix, the myofibers become deformed beyond physiological limits due to the lacking of mechanical support and impairment of a pathway of force transmission by the extracellular matrix. This leads to calculation of a drop of muscle force and if the impairment is located more towards the center of the muscle model, its effects are more pronounced. These results indicate the significance of non-myotendinous force transmission pathways.     

Aging alters contractile properties and fiber morphology in pigeon skeletal muscle

In this study, we tested the hypothesis that skeletal muscle from pigeons would display age-related alterations in isometric force and contractile parameters as well as a shift of the single muscle fiber cross-sectional area (CSA) distribution toward smaller fiber sizes. Maximal force output, twitch contraction durations and the force–frequency relationship were determined in tensor propatagialis pars biceps muscle from young 3-year-old pigeons, middle-aged 18-year-old pigeons, and aged 30-year-old pigeons. The fiber CSA distribution was determined by planimetry from muscle sections stained with hematoxylin and eosin. Maximal force output of twitch and tetanic contractions was greatest in muscles from young pigeons, while the time to peak force of twitch contractions was longest in muscles from aged pigeons. There were no changes in the force–frequency relationship between the age groups. Interestingly, the fiber CSA distribution in aged muscles revealed a greater number of larger sized muscle fibers, which was verified visually in histological images. Middle-aged and aged muscles also displayed a greater amount of slow myosin containing muscle fibers. These data demonstrate that muscles from middle-aged and aged pigeons are susceptible to alterations in contractile properties that are consistent with aging, including lower force production and longer contraction durations. These functional changes were supported by the appearance of slow myosin containing muscle fibers in muscles from middle-aged and aged pigeons. Therefore, the pigeon may represent an appropriate animal model for the study of aging-related alterations in skeletal muscle function and structure.     

Motor Force Homeostasis in Skeletal Muscle Contraction

In active biological contractile processes such as skeletal muscle contraction, cellular mitosis, and neuronal growth, an interesting common observation is that multiple motors can perform coordinated and synchronous actions, whereas individual myosin motors appear to randomly attach to and detach from actin filaments. Recent experiment has demonstrated that, during skeletal muscle shortening at a wide range of velocities, individual myosin motors maintain a force of ∼6 pN during a working stroke. To understand how such force-homeostasis can be so precisely regulated in an apparently chaotic system, here we develop a molecular model within a coupled stochastic-elastic theoretical framework. The model reveals that the unique force-stretch relation of myosin motor and the stochastic behavior of actin-myosin binding cause the average number of working motors to increase in linear proportion to the filament load, so that the force on each working motor is regulated at ∼6 pN, in excellent agreement with experiment. This study suggests that it might be a general principle to use catch bonds together with a force-stretch relation similar to that of myosin motors to regulate force homeostasis in many biological processes.     

A biomechanical model for analysis of muscle force,power output and lower jaw motion in fishes

Fish skulls are complex kinetic systems with movable components that are powered by muscles. Cranial muscles for jaw closing pull the mandible around a point of rotation at the jaw joint using a third-order lever mechanism. The present study develops a lever model for the jaw of fishes that uses muscle design and the Hill equation for nonlinear length-tension properties of muscle to calculate dynamic power output. The model uses morphometric data on skeletal dimensions and muscle proportions in order to predict behavior and force transmission mediated by lever action. The computer model calculates a range of dynamic parameters of jaw function including muscle force, torque, effective mechanical advantage, jaw velocity, bite duration, bite force, work and power. A complete list of required morphometrics is presented and a software program (MandibLever 2.0) is available for implementing lever analysis. Results show that simulations yield kinematics and timing profiles similar to actual fish feeding events. Simulation of muscle properties shows that mandibles reach their peak velocity near the start of jaw closing, peak force at the end of jaw closing, and peak power output at about 25% of the closing cycle time. Adductor jaw muscles with different mechanical designs must have different contractile properties and/or different muscle activity patterns to coordinate jaw closing. The effective mechanical advantage calculated by the model is considerably lower than the mechanical advantage estimated from morphological lever ratios, suggesting that previous studies of morphological lever ratios have overestimated force and underestimated velocity transmission to the mandible. A biomechanical model of jaw closing can be used to interpret the mechanics of a wide range of jaw mechanisms and will enable studies of the functional results of developmental and evolutionary changes in skull morphology and physiology.     

Skeletal muscle contractile protein function is preserved in human heart failure

Skeletal muscle weakness is a common finding in patients with chronic heart failure (CHF). This functional deficit cannot be accounted for by muscle atrophy alone, suggesting that the syndrome of heart failure induces a myopathy in the skeletal musculature. To determine whether decrements in muscle performance are related to alterations in contractile protein function, biopsies were obtained from the vastus lateralis muscle of four CHF patients and four control patients. CHF patients exhibited reduced peak aerobic capacity and knee extensor muscle strength. Decrements in whole muscle strength persisted after statistical control for muscle size. Thin filaments and myosin were isolated from biopsies and mechanically assessed using the in vitro motility assay. Isolated skeletal muscle thin-filament function, however, did not differ between CHF patients and controls with respect to unloaded shortening velocity, calcium sensitivity, or maximal force. Similarly, no difference in maximal force or unloaded shortening velocity of isolated myosin was observed between CHF patients and controls. From these results, we conclude that skeletal contractile protein function is unaltered in CHF patients. Other factors, such as a decrease in total muscle myosin content, are likely contributors to the skeletal muscle strength deficit of heart failure.     

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) ^1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency ³. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction ⁴. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues ⁵. This later change increases muscle stiffness ⁶. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.     

Mechanical and electrical adaptation of skeletal muscle to gravitational unloading

The physiological and biochemical properties of limb skeletal muscle have been shown to adapt to variety of experimental conditions. Among these is the microgravity encountered with spaceflight. It is adaptations accompanying skeletal muscle disuse atrophy. Foremost among these changes is a reduction in the force-generating capacity, which is presumably a direct result of decrease in fiber number and diameter. These changes suggest a spaceflight-induced reduction in muscle work capacity. The interesting finding that the reduction of the mechanical tension is not proportional to the reduction of muscle weight, fiber diameter, and concentration of contractile protein suggested that changes of electrical activity might contribute to the reduction of the contraction force in disused muscle. The purpose of our study was to assess the effects of a 7-d "dry" immersion on the contractile properties of the triceps surae muscle.     

The length dependence of muscle active force: considerations for parallel elastic properties.

The purpose of this study was to choose between two popular models of skeletal muscle: one with the parallel elastic component in parallel with both the contractile element and the series elastic component (model A), and the other in which it is in parallel with only the contractile element (model B). Passive and total forces were obtained at a variety of muscle lengths for the medial gastrocnemius muscle in anesthetized rats. Passive force was measured before the contraction (passive A) or was estimated for the fascicle length at which peak total force occurred (passive B). Fascicle length was measured with sonomicrometry. Active force was calculated by subtracting passive (A or B) force from peak total force at each fascicle or muscle length. Optimal length, that fascicle length at which active force is maximized, was 13.1 +/- 1.2 mm when passive A was subtracted and 14.0 +/- 1.1 mm with passive B (P < 0.01). Furthermore, the relationship between double-pulse contraction force and length was broader when calculated with passive B than with passive A. When the muscle was held at a long length, passive force decreased due to stress relaxation. This was accompanied by no change in fascicle length at the peak of the contraction and only a small corresponding decrease in peak total force. There is no explanation for the apparent increase in active force that would be obtained when subtracting passive A from the peak total force. Therefore, to calculate active force, it is appropriate to subtract passive force measured at the fascicle length corresponding to the length at which peak total force occurs, rather than passive force measured at the length at which the contraction begins.     

Frequency response model of skeletal muscle and its association with contractile properties of skeletal muscle

The aims of the present study were to develop a mathematical model of the skeletal muscle based on the frequency transfer function, referred to as frequency response model, and to presume the relationship between the model elements and skeletal muscle contractile properties. Twitch force in elbow flexion was elicited by applying a single electrical stimulation to the motor point of biceps brachii muscles, and then analyzed visually by the Bode gain and phase diagram of the force signal. The frequency response model was represented by a frequency transfer function consisting of five basic control elements (proportional element, dead time element, and three first-order lag elements). The model element constants were estimated by best-fitting to the Bode gain and phase diagram of the twitch force signal. The proportional constant and the dead time in the frequency response model correlated significantly with the peak torque and the latency in the actual twitch force, respectively. In addition, the time constants of the three first-order lag elements in the model correlated strongly with the contraction time and the half relaxation time in the actual twitch force. The results suggested a possibility that the individual elements in the frequency response model would reflect the biochemical and biomechanical properties in the excitation–contraction coupling process of skeletal muscle.     

Stiffness-distortion sarcomere model for muscle simulation.

A relatively simple method is presented for incorporating cross-bridge mechanisms into a muscle model. The method is based on representing force in a half sarcomere as the product of the stiffness of all parallel cross bridges and their average distortion. Differential equations for sarcomeric stiffness are derived from a three-state kinetic scheme for the cross-bridge cycle. Differential equations for average distortion are derived from a distortional balance that accounts for distortion entering and leaving due to cross-bridge cycling and for distortion imposed by shearing motion between thick and thin filaments. The distortion equations are unique and enable sarcomere mechanodynamics to be described by only a few ordinary differential equations. Model predictions of small-amplitude step and sinusoidal responses agreed well with previously described experimental results and allowed unique interpretations to be made of various response components. Similarly good results were obtained for model reproductions of force-velocity and large-amplitude step and ramp responses. The model allowed reasonable predictions of contractile behavior by taking into account what is understood to be basic muscle contractile mechanisms.     

Distribution of calcium during contraction and relaxation of crayfish skeletal muscle fibre.

A model of activation of muscle contraction has been applied to the crayfish isolated skeletal muscle fibre. The model is based on calcium diffusion and binding to specific regulatory sites in a sarcomere. Calcium ions activate interactions of contractile proteins and thus the generation of force. The model quantifies the relation between calcium released from intracellular stores and force elicited. Experimental tension records from isolated crayfish skeletal muscle fibres under voltage clamp conditions are analyzed. Model parameters were determined either via approximation of the onset of tension by the model solution or from the model based relations between the tension maximum, and depolarizing pulse length and amplitude. This allowed to determine time changes of free and bound calcium distribution in the sarcomere and the calcium release from terminal cisternae. The steady state calcium concentration at terminal cisternae showed S-shaped voltage dependence with saturation below approx. 10 mumol/l at positive membrane potentials.