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• Experimental determination of specific muscle tension in dogs based on in vivo muscle forces

Purpose Intrinsic laryngeal skeletal muscle bioenergetics, the means by which muscles produce fuel for muscle metabolism, is an understudied aspect of laryngeal physiology with direct implications for voice habilitation and rehabilitation. The purpose of this review is to describe bioenergetic pathways identified in limb skeletal muscle and introduce bioenergetic physiology as a necessary parameter for theoretical models of laryngeal skeletal muscle function. Method A comprehensive review of the human intrinsic laryngeal skeletal muscle physiology literature was conducted. Findings regarding intrinsic laryngeal muscle fiber complement and muscle metabolism in human models are summarized and exercise physiology methodology is applied to identify probable bioenergetic pathways used for voice function. Results Intrinsic laryngeal skeletal muscle fibers described in human models support the fast, high-intensity physiological requirements of these muscles for biological functions of airway protection. Inclusion of muscle bioenergetic constructs in theoretical modeling of voice training, detraining, fatigue, and voice loading have been limited. Conclusions Muscle bioenergetics, a key component for muscle training, detraining, and fatigue models in exercise science, is a little-considered aspect of intrinsic laryngeal skeletal muscle physiology. Partnered with knowledge of occupation-specific voice requirements, application of bioenergetics may inform novel considerations for voice habilitation and rehabilitation.

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.

Muscle forces are produced by repeated stereotypical actomyosin units called sarcomeres. Sarcomeres are chained into linear myofibrils spanning the entire muscle fiber. In mammalian body muscles, myofibrils are aligned laterally, resulting in their typical cross-striated morphology. Despite this detailed textbook knowledge about the adult muscle structure, it is still unclear how cross-striated myofibrils are built in vivo. Here, we investigate the morphogenesis of Drosophila abdominal muscles and establish them as an in vivo model for cross-striated muscle development. By performing live imaging, we find that long immature myofibrils lacking a periodic actomyosin pattern are built simultaneously in the entire muscle fiber and then align laterally to give mature cross-striated myofibrils. Interestingly, laser micro-lesion experiments demonstrate that mechanical tension precedes the formation of the immature myofibrils. Moreover, these immature myofibrils do generate spontaneous Ca2+-dependent contractions in vivo, which, when chemically blocked, result in cross-striation defects. Taken together, these results suggest a myofibrillogenesis model in which mechanical tension and spontaneous muscle twitching synchronize the simultaneous self-organization of different sarcomeric protein complexes to build highly regular cross-striated myofibrils spanning the length of large muscle fibers., Summary: In Drosophila, immature myofibrils are built simultaneously across an entire muscle fiber, and then self-organize in a manner dependent on spontaneous contractions and mechanical tension.

Engineered skeletal muscle tissues can be used for in vitro studies that require physiologically relevant models of native tissues. Herein, we describe the development of a three-dimensional (3D) skeletal muscle tissue that recapitulates the architectural and structural complexities of muscle within a microfluidic device. Using a 3D photo-patterning approach, we spatially confined a cell-laden gelatin network around two bio-inert hydrogel pillars, which induce uniaxial alignment of the cells and serve as anchoring sites for the encapsulated cells and muscle tissues as they form and mature. We have characterized the tissue morphology and strain profile during differentiation of the cells and skeletal muscle tissue formation by using a combination of fluorescence microscopy and computational tools. The time-dependent strain profile suggests the existence of individual cells within the gelatin matrix, which differentiated to form a multinucleated skeletal muscle tissue bundle as a function of culture time. We have also developed a method to calculate the passive tension generated by the engineered muscle tissue bundles suspended between two pillars. Finally, as a proof-of-concept we have examined the applicability of the skeletal muscle-on-chip system as a screening platform and in vitro muscle injury model. We studied the dose-dependent effect of cardiotoxin on the engineered muscle tissue architecture and its subsequent effect on the passive tension. This simple yet effective tool can be appealing for studies that necessitate the analysis of skeletal muscle structure and function, including preclinical drug discovery and development.

There is a growing demand for a system in the field of sarcopenia and diabetes research that could be used to evaluate the effects of functional food ingredients that enhance muscle mass/contractile force or muscle glucose uptake. In this study, we developed a new type of in vitro muscle incubation system that systemizes an apparatus for muscle incubation, using an electrode, a transducer, an incubator, and a pulse generator in a compact design. The new system enables us to analyze the muscle force stimulated by the electric pulses and glucose uptake during contraction and it may thus be a useful tool for analyzing the metabolic changes that occur during muscle contraction. The system may also contribute to the assessments of new food ingredients that act directly on skeletal muscle in the treatment of sarcopenia and diabetes.

Skeletal muscle has important metabolic functions but the focus of this article is to examine its ability to generate mechanical force. Adult skeletal muscle fibres are each innervated by a single branch of the axon arising from an α-motoneuron in the spinal cord. The α-motoneuron and all the fibres it innervates constitute a motor unit, and this is the functional unit of the muscle. α-Motoneurons differ in size and excitability and it is the recruitment of these cell bodies in the spinal cord that determines which fibres within the muscle are active during a movement. Correct functioning of the neuromuscular junction is clearly critical for muscle action and it is a site at which many drugs affecting muscle have their action. Here we describe also the mechanism by which skeletal muscle generates force following activation, a process known as excitation–contraction coupling and examine the contractile properties of muscle as well as describing muscle weakness and fatigue and the assessment of muscle performance in health and disease.

Defining the physiological condition of smooth muscle in more detail is the key to understanding the disease mechanism of smooth muscle defective diseases and the development of novel medical devices to help patients with these diseases. Although previous studies have detailed the physiological condition of smooth muscle contraction in gastrointestinal tracts, the precise characteristics of muscle contraction before or after stimulation have yet to be determined due to a lack of accuracy or inconsistency. Here, we obtain the passive characteristic parameters of smooth muscle and the active characteristic parameters of smooth muscle contraction from the porcine small intestine. To obtain the passive characteristic parameters of smooth muscle, we measured tensile strength in the porcine small intestine. The maximum repulsive force, 0.702 N, was measured in tensile tests. To estimate the active characteristic parameters of smooth muscle, we stimulated with acetylcholine and measured the isometric and isotoni...

Investigating the path functionality of the nerve stimulation signal and the muscle contraction is of primary importance in the study of a wide variety of pathologic conditions: neuromuscular diseases like Amyotrophic Lateral Sclerosis and Duchenne Muscular Dystrophy, as well as acute denervation and aging. Alterations of coupling between motor neuron conduction and muscle contraction can be studied in mice, comparing the muscle contraction elicited by two alternating stimulation paradigms: direct stimulation on the membrane and indirect stimulation through the nerve. The fundamental assumption behind this approach is that in a healthy model the two stimulations should lead to the same contractile response of the muscle. In this work we have searched for the pulse stimulation parameters that better resemble the physiological action potential. Applying these optimized stimulations it is then possible to design new final protocols to evaluate all the contractile parameters of muscle tissue in a wide variety of pathological models.

Skeletal muscle comprises approximately 45% of human body mass and has an essential role in metabolism and movement. It is a plastic tissue that adapts to use, disease, ageing, and malignancies. This chapter summarizes aspects of normal muscle development, growth, structure, proprioceptors, and muscle contraction. Muscle fibers are multinucleated and form from the fusion of postmitotic myoblasts. The regular, sarcomeric arrangement of proteins gives skeletal muscle its characteristic striated pattern. In contrast to cardiac muscle, skeletal muscles are composed of fibers with different biochemical and physiological properties, the proportions of which vary between muscles. Muscle has an extensive vascular network and all fibers are innervated by one nerve. However, the axon of the motor neuron is branched and innervates a number of fibers. The anterior horn cell, axon, and muscle fibers it innervates constitute a functional motor unit. The axon interacts with the muscle fiber at specialized regions known as neuromuscular junctions allowing an action potential to depolarize the muscle fiber membrane and induce the release of calcium, resulting in contraction.

Tissue-engineered skeletal muscle can serve as a physiological model of natural muscle and a potential therapeutic vehicle for rapid repair of severe muscle loss and injury. Here, we describe a platform for engineering and testing highly functional biomimetic muscle tissues with a resident satellite cell niche and capacity for robust myogenesis and self-regeneration in vitro. Using a mouse dorsal window implantation model and transduction with fluorescent intracellular calcium indicator, GCaMP3, we nondestructively monitored, in real time, vascular integration and the functional state of engineered muscle in vivo. During a 2-wk period, implanted engineered muscle exhibited a steady ingrowth of blood-perfused microvasculature along with an increase in amplitude of calcium transients and force of contraction. We also demonstrated superior structural organization, vascularization, and contractile function of fully differentiated vs. undifferentiated engineered muscle implants. The described in vitro and in vivo models of biomimetic engineered muscle represent enabling technology for novel studies of skeletal muscle function and regeneration.

For in vitro tissue engineering of skeletal muscle, alignment and fusion of the cultured skeletal muscle cells are required. Although the successful alignment of skeletal muscle cells cultured in collagen gel has been reported using a mechanical force, other means of aligning cultured skeletal muscle cells have not been described. However, skeletal muscle cells cultured in a two-dimensional dish have been reported to align in a uniform direction when electrically stimulated. The purpose of this study is to determine if skeletal muscle cells cultured three-dimensionally in collagen gels can be aligned by an electrical load. By adding direct current to cells of the C2C12 skeletal muscle cell line cultured in collagen gel, it was possible to align C2C12 cells in a similar direction. However, the ratio of alignment was better when mechanical force was used as the means of alignment. Thus for tissue engineering of skeletal muscle cells, electrical stimulation may be useful as a supplementary method.

The field of tissue engineering involves design of high-fidelity tissue substitutes for predictive experimental assays in vitro and cell-based regenerative therapies in vivo. Design of striated muscle tissues, such as cardiac and skeletal muscle, has been particularly challenging due to a high metabolic demand and complex cellular organization and electromechanical function of the native tissues. Successful engineering of highly functional striated muscles may thus require creation of biomimetic culture conditions involving medium perfusion, electrical and mechanical stimulation. When optimized, these external cues are expected to synergistically and dynamically activate important intracellular signaling pathways leading to accelerated muscle growth and development. This review will discuss the use of different types of tissue culture bioreactors aimed at providing conditions for enhanced structural and functional maturation of engineered striated muscles.

Little is known about the contraction and relaxation properties of fetal skeletal muscle, and measurements thus far have been made with non-human mammalian muscle. Data on human fetal skeletal muscle contraction are lacking, and there are no published reports on the kinetics of either fetal or adult human skeletal muscle myofibrils. Understanding the contractile properties of human fetal muscle would be valuable in understanding muscle development and a variety of muscle diseases that are associated with mutations in fetal muscle sarcomere proteins. Therefore, we characterised the contractile properties of developing human fetal skeletal muscle and compared them to adult human skeletal muscle and rabbit psoas muscle. Electron micrographs showed human fetal muscle sarcomeres are not fully formed but myofibril formation is visible. Isolated myofibril mechanical measurements revealed much lower specific force, and slower rates of isometric force development, slow phase relaxation, and fast phase relaxation in human fetal when compared to human adult skeletal muscle. The duration of slow phase relaxation was also significantly longer compared to both adult groups, but was similarly affected by elevated ADP. F-actin sliding on human fetal skeletal myosin coated surfaces in in vitro motility (IVM) assays was much slower compared with adult rabbit skeletal myosin, though the Km(app) (apparent (fitted) Michaelis-Menten constant) of F-actin speed with ATP titration suggests a greater affinity of human fetal myosin for nucleotide binding. Replacing ATP with 2 deoxy-ATP (dATP) increased F-actin speed for both groups by a similar amount. Titrations of ADP into IVM assays produced a similar inhibitory affect for both groups, suggesting ADP binding may be similar, at least under low load. Together, our results suggest slower but similar mechanisms of myosin chemomechanical transduction for human fetal muscle that may also be limited by immature myofilament structure.

There exists several numerical approaches to describe the active contractile behaviour of skeletal muscles. These models range from simple one-dimensional to more advanced three-dimensional ones; especially, three-dimensional models take up the cause of describing complex contraction modes in a realistic way. However, the validation of such concepts is challenging, as the combination of geometry, material and force characteristics is so far not available from the same muscle. To this end, we present in this study a comprehensive data set of the rabbit soleus muscle consisting of the muscles' characteristic force responses (active and passive), its three-dimensional shape during isometric, isotonic and isokinetic contraction experiments including the spatial arrangement of muscle tissue and aponeurosis-tendon complex, and the fascicle orientation throughout the whole muscle at its optimal length. In this way, an extensive data set is available giving insight into the three-dimensional geometry of the rabbit soleus muscle and, further, allowing to validate three-dimensional numerical models.

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Skeletal muscle can adapt to increased mechanical loads by undergoing hypertrophy. Transient reductions in whole muscle force production have been reported during the onset of hypertrophy, but contractile changes in individual muscle fibers have not been previously studied. Additionally, the extracellular matrix (ECM) stores and transmits forces from muscle fibers to tendons and bones, and determining how the ECM changes during hypertrophy is important in understanding the adaptation of muscle tissue to mechanical loading. Using the synergist ablation model, we sought to measure changes in muscle fiber contractility, collagen content, and cross-linking, and in the expression of several genes and activation of signaling proteins that regulate critical components of myogenesis and ECM synthesis and remodeling during muscle hypertrophy. Tissues were harvested 3, 7, and 28 days after induction of hypertrophy, and nonoverloaded rats served as controls. Muscle fiber specific force (sFo), which is the maximum isometric force normalized to cross-sectional area, was reduced 3 and 7 days after the onset of mechanical overload, but returned to control levels by 28 days. Collagen abundance displayed a similar pattern of change. Nearly a quarter of the transcriptome changed over the course of overload, as well as the activation of signaling pathways related to hypertrophy and atrophy. Overall, this study provides insight into fundamental mechanisms of muscle and ECM growth, and indicates that although muscle fibers appear to have completed remodeling and regeneration 1 mo after synergist ablation, the ECM continues to be actively remodeling at this time point.NEW & NOTEWORTHY This study utilized a rat synergist ablation model to integrate changes in single muscle fiber contractility, extracellular matrix composition, activation of important signaling pathways in muscle adaption, and corresponding changes in the muscle transcriptome to provide novel insight into the basic biological mechanisms of muscle fiber hypertrophy.

Analysis of muscle reconstruction research is carried out in the article, and a model of force transmission processes from myofilaments to tendon is proposed. The model allows observe the sequence of the processes occurring in muscle during concentric and eccentric contraction mode.

Tissue-engineered skeletal muscle has the promise to be a tool for studying physiology, screening muscle-active drugs, and clinical replacement of damaged muscle. To maximize the potential benefits of engineered muscle, it is important to understand the factors required for tissue formation and how these affect muscle function. In this review, we evaluate how biomaterials, cell source, media components, and bioreactor interventions impact muscle function and phenotype.

Permeabilized individual skeletal muscle fibers offer the opportunity to evaluate contractile behavior in a system that is greatly simplified, yet physiologically relevant. Here we describe the steps required to prepare, permeabilize and preserve small samples of skeletal muscle. We then detail the procedures used to isolate individual fiber segments and attach them to an experimental apparatus for the purpose of controlling activation and measuring force generation. We also describe our technique for estimating the cross-sectional area of fiber segments. The area measurement is necessary for normalizing the absolute force to obtain specific force, a measure of the intrinsic force-generating capability of the contractile system.

In order to investigate the molecular and cellular mechanisms of muscle regeneration an experimental injury model is required. Advantages of eccentric contraction-induced injury are that it is a controllable, reproducible, and physiologically relevant model to cause muscle injury, with injury being defined as a loss of force generating capacity. While eccentric contractions can be incorporated into conscious animal study designs such as downhill treadmill running, electrophysiological approaches to elicit eccentric contractions and examine muscle contractility, for example before and after the injurious eccentric contractions, allows researchers to circumvent common issues in determining muscle function in a conscious animal (e.g., unwillingness to participate). Herein, we describe in vitro and in vivo methods that are reliable, repeatable, and truly maximal because the muscle contractions are evoked in a controlled, quantifiable manner independent of subject motivation. Both methods can be used to initiate eccentric contraction-induced injury and are suitable for monitoring functional muscle regeneration hours to days to weeks post-injury.

article i nfo Introduction: To evaluate the viability of a muscle tissue, it is essential to measure the tissue's contractile performance as well as to control its structure. Accurate contractility data can aid in development of more ef- fective and safer drugs. This can be accomplished with a robust in vitro contractility assay applicable to var- ious types of muscle tissue. Methods: The devices developed in this work were based on the muscular thin film (MTF) technology, in which an elastic film is manufactured with a 2D engineered muscle tissue on one side. The tissue template is made by patterning extracellular matrix with microcontact printing. When muscle cells are seeded on the film, they self-organize with respect to the geometric cues in the matrix to form a tissue. Results: Several assays based on the "MTF on a chip" technology are demonstrated. One such assay incorporates the contractility assay with striated muscle into a fluidic channel. Another assay platform in- corporates the MTFs in a multi-well plate, which is compatible with automated data collection and analysis. Fi- nally, we demonstrate the possibility of analyzing contractility of both striated and smooth muscle simultaneously on the same chip. Discussion: In this work, we assembled an ensemble of contractility assays for striated and smooth muscle based on muscular thin films. Our results suggest an improvement over current methods and an alternative to isolated tissue preparations. Our technology is amenable to both primary har- vests cells and cell lines, as well as both human and animal tissues.

The field of skeletal muscle tissue engineering is currently hampered by the lack of methods to form large muscle constructs composed of dense, aligned, and mature myofibers and limited understanding of structure-function relationships in developing muscle tissues. In our previous studies, engineered muscle sheets with elliptical pores ("muscle networks") were fabricated by casting cells and fibrin gel inside elastomeric tissue molds with staggered hexagonal posts. In these networks, alignment of cells around the elliptical pores followed the local distribution of tissue strains that were generated by cell-mediated compaction of fibrin gel against the hexagonal posts. The goal of this study was to assess how systematic variations in pore elongation affect the morphology and contractile function of muscle networks. We found that in muscle networks with more elongated pores the force production of individual myofibers was not altered, but the myofiber alignment and efficiency of myofiber formation were significantly increased yielding an increase in the total contractile force despite a decrease in the total tissue volume. Beyond a certain pore length, increase in generated contractile force was mainly contributed by more efficient myofiber formation rather than enhanced myofiber alignment. Collectively, these studies show that changes in local tissue geometry can exert both direct structural and indirect myogenic effects on the functional output of engineered muscle. Different hydrogel formulations and pore geometries will be explored in the future to further augment contractile function of engineered muscle networks and promote their use for basic structure-function studies in vitro and, eventually, for efficient muscle repair in vivo.

Chronic skeletal muscle stretch typically increases serial muscle fiber sarcomere number. Since serial sarcomere number correlates with functional excursion in normal muscle, observed changes in sarcomere number are often extrapolated to their new assumed function. However, this has not been well demonstrated experimentally. Thus, we measured the functional properties of muscles stretched due to tendon transfer surgery. Muscle active and passive length-tension curves were measured 1 week and 4 weeks after surgery, and then each muscle was further examined to determine structural adaptation as well as single fiber and fiber bundle passive mechanical properties. We found a disconnect between the functional and structural muscle properties. Specifically, muscle excursion was significantly lower in the transferred muscle compared to controls, even though serial sarcomere number had increased. Furthermore, maximum tetanic tension was significantly reduced, though the two groups had similar physiological cross sectional areas. Passive tension increased in the transferred muscle, which was deemed to be due to proliferation of extracellular matrix. These data are the first to report that muscle morphological adaptation after chronic stretch does not accurately predict the muscle’s functional properties. These data have significant implications for examining muscle physiological properties under surgical interventions.

The use of rodents to model neuromuscular diseases necessitates assessment of neuromuscular function to monitor disease progression. Muscle function can be assessed by determining muscle force, and the contraction's contractile and fatigue characteristics. Assessment of motor units gives a measure of motoneuron health. Thus, assessment of these parameters can reveal the degree and nature of neuromuscular pathology. A reduction in muscle force may result either from loss of motoneurons and a concomitant denervation of muscles or as a result of primary muscle pathology. Estimation of the number of functional motor units may identify whether the deficit is neural in origin. Here, we give a detailed description of the assessment of muscle force, contractile characteristics, and muscle fatigue, as well as a method that gives a direct and accurate readout on the number of motor units in individual mouse hindlimb muscles in mice-now widely used to model a variety of neuromuscular disorders. Curr. Protoc. Mouse Biol. 2:89-101 © 2012 by John Wiley & Sons, Inc.

Biomechanical models of whole muscles commonly used in simulations of musculoskeletal function and movement typically assume that the muscle generates force as a scaled-up muscle fiber. However, muscles are comprised of motor units that have different intrinsic properties and that can be activated at different times. This study tested whether a muscle model comprised of motor units that could be independently activated resulted in more accurate predictions of force than traditional Hill-type models. Forces predicted by the models were evaluated by direct comparison with the muscle forces measured in situ from the gastrocnemii in goats. The muscle was stimulated tetanically at a range of frequencies, muscle fiber strains were measured using sonomicrometry, and the activation patterns of the different types of motor unit were calculated from electromyographic recordings. Activation patterns were input into five different muscle models. Four models were traditional Hill-type models with different intrinsic speeds and fiber-type properties. The fifth model incorporated differential groups of fast and slow motor units. For all goats, muscles and stimulation frequencies the differential model resulted in the best predictions of muscle force. The in situ muscle output was shown to depend on the recruitment of different motor units within the muscle.

The development of engineered skeletal muscle would provide a viable tissue for replacement and repair of muscle damaged by disease or injury. Our current tissue-engineering methods result in three-dimensional (3D) muscle constructs that generate tension but do not advance phenotypically beyond neonatal characteristics. To develop to an adult phenotype, innervation and vascularization of the construct must occur. In this study, 3D muscle constructs were implanted into the hindlimb of a rat, along the sciatic nerve, with the sural nerve isolated, transected and sutured to the construct to encourage innervation. Aortic ring anchors were sutured to the tendons of the biceps femoris muscle so that the construct would move dynamically with the endogenous muscle. After 1 week in vivo, the constructs were explanted, evaluated for force production and stained for muscle, nerve and collagen markers. Implanted muscle constructs showed a developing capillary system, an epimysium-like outer layer of connective tissue and an increase in myofibre content. The beginning of α-bungarotoxin clustering suggests that neuromuscular junctions (NMJs) could form on the implanted muscle, given more time in vivo. Additionally, the constructs increased maximum isometric force from 192 ± 41 μN to 549 ± 103 μN (245% increase) compared to in vitro controls, which increased from 276 ± 23 μN to 329 ± 27μN (25% increase). These findings suggest that engineered muscle tissue survives 1 week of implantation and begins to develop the necessary interfaces needed to advance the phenotype toward adult muscle. However, in terms of force production, the muscle constructs need longer implantation times to fully develop an adult phenotype.

The genetic advantages of Drosophila make it a very appealing choice for investigating muscle development, muscle physiology and muscle protein structure and function. To take full advantage of this model organism, it has been vital to develop isolated Drosophila muscle preparations that can be mechanically evaluated. We describe techniques to isolate, prepare and mechanically analyze skinned muscle fibers from two Drosophila muscle types, the indirect flight muscle and the jump muscle. The function of the indirect flight muscle is similar to vertebrate cardiac muscle, to generate power in an oscillatory manner. The indirect flight muscle is ideal for evaluating the influence of protein mutations on muscle and cross-bridge stiffness, oscillatory power, and deriving cross-bridge rate constants. Jump muscle physiology and structure are more similar to skeletal vertebrate muscle than indirect flight muscle, and it is ideal for measuring maximum shortening velocity, force-velocity characteristics and steady-state power generation.

With a remarkable plasticity, skeletal muscle adapts to an altered functional demand. Muscle angio-adaptation can either involve the growth or the regression of capillaries as respectively observed in response to endurance training or muscle unloading. Whereas the molecular mechanisms that regulate exercise-induced muscle angiogenesis have been extensively studied, understanding how muscle unloading can in contrast lead to capillary regression has received very little attention. Here we have investigated the consequences of a 9 day time course hindlimb unloading on both capillarization and expression of angio-adaptive molecules in two different rat skeletal muscles. Both soleus and plantaris muscles were atrophied similarly. In contrast, our results have shown different angio-adaptive patterns between these two muscles. Capillary regression occurred only in the soleus, a slow-twitch and oxidative postural muscle. Conversely, the level of capillarization was preserved in the plantaris, a fast-twitch and glycolytic muscle. We have also measured the time course protein expression of key pro- and anti-angiogenic signals (VEGF-A, VEGF-B, VEGF-R2, TSP-1). Our results have revealed that the angio-adaptive response to unloading was muscle-type specific, and that an integrated balance between pro- and anti-angiogenic signals plays a determinant role in regulating this process. In conclusion, we have brought new evidence that measuring the ratio between pro- and anti-angiogenic signals in order to evaluate muscle angio-adaptation was a more accurate approach than analysing the expression of molecular factors taken individually.

From the muscles that control the blink of your eye to those that allow you to walk, the basic architecture of muscle is the same: muscles consist of bundles of the unit muscle cell, the muscle fiber. The unique morphology of the individual muscle fiber is dictated by the functional demands necessary to generate and withstand the forces of contraction, which in turn leads to movement. Contractile muscle fibers are elongated, syncytial cells, which interact with both the nervous and skeletal systems to govern body motion. In this review, we focus on three key cell-cell and cell-matrix contact processes, that are necessary to create this exquisitely specialized cell: cell fusion, cell elongation, and establishment of a myotendinous junction. We address these processes by highlighting recent findings from the Drosophila model system.

Muscle is generally divided into three subtypes-skeletal, cardiac, and smooth-but because this edition focuses on the musculoskeletal system, this article concentrates on skeletal muscle. We review ultrastructure and function and then look at the latest scientific ideas concerning the physiological basis of muscle contraction. It is important to appreciate the different muscle types and how they act with respect to muscle growth and adaptation. Finally, what happens to muscle cells when they are damaged and the reparative response is considered.

Summary Oral function of the patient improves and muscle function becomes better after prosthetic treatment. Various phenomena during this process have now been clear through basic experiments. Weakened muscle tissue before dental treatment is induced by mechanical stress after the therapy and this stimulation results in expression of various genes. Then various cascades are switched on and muscle function recovers in accordance with mechanism of muscle hypertrophy. Thus involvement of the muscle stem cells, satellite cell and muscle side population (SP) cell has been come into knowledge for muscle hypertrophy. Again alterations in characteristics of muscle fibers associated with changes in function of oral field makes clear existence of remodeling of the muscle tissue, which carries the functional role. Based on results of these researches, dental treatment is proved to give rise to anti-aging for oral function.

A quantitative understanding of the bulk excitability of skeletal muscle tissues is important for the design of muscle tissue bioreactor systems, implantable muscle stimulators, and other systems where electrical pulses are employed to elicit contractions in muscle tissue both in vitro and in vivo. The purpose of the present study is to systematically compare the excitability of mammalian (rat) skeletal muscle under a range of conditions (including neonatal development, denervation, and chronic in vivo stimulation of denervated muscle) and of self-organized muscle tissue constructs engineered in vitro from both primary cells and cell lines. Excitability is represented by rheobase (R(50), units = V/mm) and chronaxie (C(50), units = microseconds) values, with lower values for each indicating greater excitability. Adult skeletal muscle is the most excitable (R(50) ~ 0.29, C(50) ~ 100); chronically denervated whole muscles (R(50) ~ 2.54, C(50) ~ 690) and muscle engineered in vitro from cell lines (C2C12 + 10T1/2) (R(50) ~ 1.93, C(50) ~ 416) have exceptionally low excitability; muscle engineered in vitro from primary myocytes (R(50) ~ 0.99, C(50) ~ 496) has excitability similar to that of day 14 neonatal rat muscle (R(50) ~ 0.65, C(50) ~ 435); stimulated-denervated muscles retain excellent excitability when chronically electrically stimulated (R(50) ~ 0.40, C(50) ~ 100); and neonatal rat muscle excitability improves during the first 6 weeks of development, steadily approaching that of adult muscle.

The ability for muscle to repeatedly generate force is limited by fatigue. The cellular mechanisms behind muscle fatigue are complex and potentially include breakdown at many points along the excitation-contraction pathway. In this paper we construct a mathematical model of the skeletal muscle excitation-contraction pathway based on the cellular biochemical events that link excitation to contraction. The model includes descriptions of membrane voltage, calcium cycling and crossbridge dynamics and was parameterised and validated using the response characteristics of mouse skeletal muscle to a range of electrical stimuli. This model was used to uncover the complexities of skeletal muscle fatigue. We also parameterised our model to describe force kinetics in fast and slow twitch fibre types, which have a number of biochemical and biophysical differences. How these differences interact to generate different force/fatigue responses in fast- and slow- twitch fibres is not well understood and we used our modelling approach to bring new insights to this relationship.

Skeletal muscles present a non-cross-bridge increase in sarcomere stiffness and tension on Ca2+ activation, referred to as static stiffness and static tension, respectively. It has been hypothesized that this increase in tension is caused by Ca2+-dependent changes in the properties of titin molecules. To verify this hypothesis, we investigated the static tension in muscles containing different titin isoforms. Permeabilized myofibrils were isolated from the psoas, soleus, and heart ventricle from the rabbit, and tested in pCa 9.0 and pCa 4.5, before and after extraction of troponin C, thin filaments, and treatment with the actomyosin inhibitor blebbistatin. The myofibrils were tested with stretches of different amplitudes in sarcomere lengths varying between 1.93 and 3.37 μm for the psoas, 2.68 and 4.21 μm for the soleus, and 1.51 and 2.86 μm for the ventricle. Using gel electrophoresis, we confirmed that the three muscles tested have different titin isoforms. The static tension was present in psoas and soleus myofibrils, but not in ventricle myofibrils, and higher in psoas myofibrils than in soleus myofibrils. These results suggest that the increase in the static tension is directly associated with Ca2+-dependent change in titin properties and not associated with changes in titin-actin interactions.

Muscle fiber tension and sarcomere length play critical roles in regulating muscle functions and adaptations under pathological conditions. However, methods are lacking to quantify these two variables simultaneously in vivo. A novel force microscope was developed with the unique capabilities of estimating muscle fiber tension and acquiring sarcomere images simultaneously in vivo. The force microscope consisting of a custom microscopic imaging system and a force sensor was used to quantify in vivo sarcomere length, muscle fiber tension and stress of the tibialis cranialis muscle at plantar-flexed and dorsi-flexed positions from 11 rat hind limbs. Results showed that sarcomere images and fiber tension could be measured together in vivo with significantly higher muscle fiber tension and stress and longer sarcomere length at the plantar-flexed position when compared to their counterparts at the dorsi-flexed position. The fiber tension estimated using the force microscope had close agreement with the direct measurements of the fiber tension. The present force microscope with simultaneous characterizations of fiber tension and sarcomere imaging provides us a useful in vivo tool to investigate the roles of muscle tension in regulating sarcomere and muscle fiber functions under physiological and pathological conditions.

Muscle weakness is an important phenotype of many diseases that is linked to impaired locomotion and increased mortality. The force that a muscle can generate is determined predominantly by muscle size, fiber type and the excitation-contraction coupling process. Here we describe methods for the histological assessment of whole muscle to determine fiber cross-sectional area and fiber type, determination of changes in myocyte size using C2C12 cells, in vivo functional tests and measurement of contractility in dissected whole muscles. The extensor digitorum longus and soleus muscles are ideally suited for whole-muscle contractility, and dissection of these muscles is described.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

The development of a functional three-dimensional model of human skeletal muscle tissue could accelerate progress towards new and personalized treatments for skeletal muscle disorders.

Rigorous study of the associations between urethral structural anatomy and biomechanical function is necessary to advance the understanding of the development, progression, and treatment of urethral pathologies. An ex vivo model was utilized to define the relative biomechanical contributions of the active (muscle) elements of the female urethra relative to its passive (noncontractile) elements. Whole urethras from female, adult rats were tested under a range of applied intraluminal pressures (0 to 20 mmHg) as a laser micrometer simultaneously measured midurethral outer diameter. Active tissue characterization was performed during induced contraction of either smooth muscle alone ( Nω-nitro-l-arginine, phenylephrine), striated muscle alone (sodium nitroprusside, atropine, hexamethonium, acetylcholine), or during collective activation of both muscles ( Nω-nitro-l-arginine, phenylephrine, acetylcholine). The subsequent collection of paired passive biomechanical responses permitted the determination of parameters related to intrinsic muscle contractile function. Activation of each muscle layer significantly influenced the biomechanical responses of the tissue. Measures of muscle responsiveness over a wide range of sustained opposing pressures indicated that an activated striated muscle component was approximately one-third as effective as activated smooth muscle in resisting tissue deformation. The maximum circumferential stress generated by the striated muscle component under these conditions was also determined to be approximately one-third of that generated by the smooth muscle (748 ± 379 vs. 2,229 ± 409 N/m2). The experiments quantitatively reveal the relative influence of the intrinsic urethral smooth and striated muscle layers with regard to their effect on the mechanical properties and maximum functional responses of the urethra to applied intralumenal stresses in the complete absence of extrinsic influences.

Assessment of skeletal muscle function is an integral part of clinical trials of pharmacology and other interventions in patients with neuromuscular disorders. Muscle function can be assessed on multiple levels ranging from the whole organism to the molecular level. At the level of the organism or person, timed functional tests such as a 30-m walk or rising from a chair can be used. At the level of the organ or muscle, strength and power can be measured using a variety of techniques including manual muscle testing, hand-held dynamometry, maximum voluntary isometric contraction measurement, and isokinetic testing. At the cellular level, the skinned single muscle fiber preparation may be used to measure cellular force and power production and contractile velocity. Finally, at the molecular level, the in vitro motility assay can be used to measure the translation velocity of actin filaments on myosin. This article will present a discussion of the usefulness of the cellular approach in the evaluation of patients with neuromuscular disease. In neuromuscular disorders, cellular muscle physiology may be directly or indirectly impaired. Thus, it is important to have a tool to directly assess the degree of impairment and the effect of rehabilitation, pharmacologic, and gene therapy interventions on that impairment. In muscle diseases, muscle physiology is directly affected. The manner in which muscle physiology is affected depends on whether the muscle protein abnormality is in the extracellular matrix (laminin), sarcolemma (dystrophin, sarcoglycan, dysferlin), sarcomere (actin, myosin, tropomyosin, titin), nuclear membrane (lamin A/C, emerin), or in energy metabolism pathways (glycogenoses, mitochondrial disorders). In neuromuscular disorders where skeletal muscle is indirectly affected by pathology in the anterior horn cell, peripheral nerve, or neuromuscular junction, secondary changes in muscle physiology may occur. The

Although it is well established that patients suffering from malaria experience skeletal muscle problems (contracture, aches, fatigue, weakness), detailed studies have not been performed to investigate changes in the contractile function and biochemical properties of intact and skinned skeletal muscles of mammals infected with malaria. To this end, we investigated such features in the extensor digitorium longus (EDL, fast-twitch, glyocolytic) and in the soleus (SOL, slow-twitch, oxidative) muscles from mice infected with Plasmodium berghei. We first studied maximal tetanic force (T(max)) produced by intact control and malaria-infected muscles before, during and after fatigue. Triton-skinned muscle fibres were isolated from these muscles and used to determine isometric contractile features as well as a basic biochemical profile as analysed by silver-enhanced SDS-PAGE. We found that the T(max) of intact muscles and the maximal Ca2+-activated force (F(max)) of Triton-skinned muscle fibres were reduced by approximately 50% in malarial muscles. In addition, the contractile proteins of Triton-skinned muscle fibres from malarial muscles were significantly less sensitive to Ca2+. Biochemical analysis revealed that there was a significant loss of essential contractile proteins (e.g. troponins and myosin) in Triton-skinned muscle fibres from malarial muscles as compared to controls. The biochemical alterations (i.e., reduction of essential contractile proteins) seem to explain well the functional modifications resolved in both intact muscles and Triton-skinned muscle fibres and may provide a suitable paradigm for the aetiology of muscle symptoms associated with malaria.

In vivo 31P-MRS investigations have been widely used in small animals to study skeletal muscle function under normal and pathological conditions. Paradoxically in these studies, the benefit provided by 31P-MRS in terms of non-invasiveness is lost because of the utilization of experimental setups that integrate invasive devices for inducing muscle contractions and for measuring mechanical performance. These traditional methodologies, which require surgical preparations, have obvious limitations regarding repeatability in the same animal. The purpose of this review is to highlight the technical aspects of the in vivo MR investigations of skeletal muscle function in small animal models. We will more particularly address the issue related to the invasiveness of different procedures used so far in order to show finally that a further step into non-invasiveness can be achieved, in particular with the support of muscle functional 1H-MRI.

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.

Force is transmitted from muscle fiber to bone via several pathways: (1) via the tendons (i.e. myotendinous force transmission), (2) via intermuscular connective tissue to adjacent muscles (i.e. intermuscular myofascial force transmission), (3) via structures other than muscles (i.e. extramuscular myofascial force transmission). In vivo, the position of a muscle relative to adjacent muscles changes due to differences in moment arm between synergists as well as due to the fact that some muscles span only one joint and other muscles more than one joint. The position of a muscle relative to non-muscular structures within a compartment is altered with each change of the length of the muscle.The aim of this article is to describe recent experimental results, as well as some new experimental data, that have elucidated the role of muscle relative position on force transmission from muscle. Furthermore, relevant literature is discussed, taking into consideration these new insights of muscle functioning. It is concluded that the position of a muscle relative to surrounding tissues is a major co-determinant of isometric muscle force. For muscles operating within their in vivo context of connective tissue, such position effects should be taken into account.

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions. The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release. The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.

In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers can also be used in measuring muscle injury, such as increased creatine kinase levels in the blood, but these are not always correlated with loss in muscle function (i.e. loss of force production). This is even true of histological findings from animals, which provide a “direct measure” of damage, but do not account for loss of function. The most comprehensive measure of the overall health of the muscle is contractile force. To date, animal models testing contractile force have been limited to the muscle groups moving the ankle. Here we describe an in vivo animal model for the quadriceps, with abilities to measure torque, produce a reliable muscle injury, and follow muscle recovery within the same animal over time. We also describe a second model used for direct measurement of force from an isolated quadriceps muscle in situ.