Altered muscle force and stiffness of skeletal muscles in alpha-sarcoglycan-deficient mice

Alpha-sarcoglycan (ASG) is a transmembrane protein of the dystrophin-associated complex, and absence of ASG causes limb-girdle muscular dystrophy. We hypothesize that disruption of the sarcoglycan complex may alter muscle extensibility and disrupt the coupling between passive transverse and axial contractile elements in the diaphragm. We determined the length-tension relationships of the diaphragm of young ASG-deficient mice and their controls during uniaxial and biaxial loading. We also determined the isometric contractile properties of the diaphragm muscles from mutant and normal mice in the absence and presence of passive transverse stress. We found that the diaphragm muscles of the null mutants for the protein ASG show 1) significant decrease in muscle extensibility in the directions of the muscle fibers and transverse to fibers, 2) significant reductions in force-generating capacity, and 3) significant reductions in coupling between longitudinal and transverse properties. Thus these findings suggest that the sarcoglycan complex serves a mechanical function in the diaphragm by contributing to muscle passive stiffness and to the modulation of the contractile properties of the muscle.     

Isoform diversity of giant proteins in relation to passive and active contractile properties of rabbit skeletal muscles

The active and passive contractile performance of skeletal muscle fibers largely depends on the myosin heavy chain (MHC) isoform and the stiffness of the titin spring, respectively. Open questions concern the relationship between titin-based stiffness and active contractile parameters, and titin's importance for total passive muscle stiffness. Here, a large set of adult rabbit muscles (n = 37) was studied for titin size diversity, passive mechanical properties, and possible correlations with the fiber/MHC composition. Titin isoform analyses showed sizes between approximately 3300 and 3700 kD; 31 muscles contained a single isoform, six muscles coexpressed two isoforms, including the psoas, where individual fibers expressed similar isoform ratios of 30:70 (3.4:3.3 MD). Gel electrophoresis and Western blotting of two other giant muscle proteins, nebulin and obscurin, demonstrated muscle type-dependent size differences of < or =70 kD. Single fiber and single myofibril mechanics performed on a subset of muscles showed inverse relationships between titin size and titin-borne tension. Force measurements on muscle strips suggested that titin-based stiffness is not correlated with total passive stiffness, which is largely determined also by extramyofibrillar structures, particularly collagen. Some muscles have low titin-based stiffness but high total passive stiffness, whereas the opposite is true for other muscles. Plots of titin size versus percentage of fiber type or MHC isoform (I-IIB-IIA-IID) determined by myofibrillar ATPase staining and gel electrophoresis revealed modest correlations with the type I fiber and MHC-I proportions. No relationships were found with the proportions of the different type II fiber/MHC-II subtypes. Titin-based stiffness decreased with the slow fiber/MHC percentage, whereas neither extramyofibrillar nor total passive stiffness depended on the fiber/MHC composition. In conclusion, a low correlation exists between the active and passive mechanical properties of skeletal muscle fibers. Slow muscles usually express long titin(s), predominantly fast muscles can express either short or long titin(s), giving rise to low titin-based stiffness in slow muscles and highly variable stiffness in fast muscles. Titin contributes substantially to total passive stiffness, but this contribution varies greatly among muscles.     

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.     

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.     

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.     

Passive mechanics of muscle tendinous junction of canine diaphragm.

The diaphragmatic muscle tendon is a biaxially loaded junction in vivo. Stress-strain relations along and transverse to the fiber directions are important in understanding its mechanical properties. We hypothesized that 1) the central tendon possesses greater passive stiffness than adjacent muscle, 2) the diaphragm muscle is anisotropic, whereas the central tendon near the junction is essentially isotropic, and 3) a gradient in passive stiffness exists as one approaches the muscle-tendinous junction (MTJ). To investigate these hypotheses, we conducted uniaxial and biaxial mechanical loading on samples of the MTJ excised from the midcostal region of dog diaphragm. We measured passive length-tension relationships of the muscle, tendon, and MTJ in the direction along the muscle fibers as well as transverse to the fibers. The MTJ was slack in the unloaded state, resulting in a J-shaped passive tension-strain curve. Generally, muscle strain was greater than that of MTJ, which was greater than tendon strain. In the muscular region, stiffness in the direction transverse to the fibers is much greater than that along the fibers. The central tendon is essentially inextensible in the direction transverse to the fibers as well as along the fibers. Our data demonstrate the existence of more pronounced anisotropy in the muscle than in the tendon near the junction. Furthermore, a gradient in muscle stiffness exists as one approaches the MTJ, consistent with the hypothesis of continuous passive stiffness across the MTJ.     

Force transmission, compliance, and viscoelasticity are altered in the alpha7-integrin-null mouse diaphragm

₇₁ integrin is a transmembrane structural and receptor protein of skeletal muscles, and the absence of ₇-integrin causes muscular dystrophy. We hypothesized that the absence of ₇-integrin alters compliance and viscoelasticity and disrupts the mechanical coupling between passive transverse and axial contractile elements in the diaphragm. In vivo the diaphragm is loaded with pressure, and therefore axial and transverse length-tension relationships are important in assessing its function. We determined diaphragm passive length-tension relationships and the viscoelastic properties of its muscle in 1-month-old ₇-integrin-null mice and age-matched controls. Furthermore, we measured the isometric contractile properties of the diaphragm from mutant and normal mice in the absence and presence of passive force applied in the transverse direction to fibers in 1-month-old and 5-month-old mutant mice. We found that compared with controls, the diaphragm direction of ₇-integrin-null mutants showed 1) a significant decrease in muscle extensibility in 1-year-old mice, whereas muscle extensibility increased in the 1-month-old mice; 2) altered muscle viscoelasticity in the transverse direction of the muscle fibers of 1-month-old mice; 3) a significant increase in force-generating capacity in the diaphragms of 1-month-old mice, whereas in 5-month-old mice muscle contractility was depressed; and 4) significant reductions in mechanical coupling between longitudinal and transverse properties of the muscle fibers of 1-month-old mice. These findings suggest that ₇-integrin serves an important mechanical function in the diaphragm by contributing to passive compliance, viscoelasticity, and modulation of its muscle contractile properties. muscular dystrophy; respiratory muscles; transmembrane proteins     

The role of transmembrane proteins on force transmission in skeletal muscle

Lateral transmission of force from myofibers laterally to the surrounding extracellular matrix (ECM) via the transmembrane proteins between them is impaired in old muscles. Changes in geometrical and mechanical properties of ECM of skeletal muscle do not fully explain the impaired lateral transmission with aging. The objective of this study was to determine the role of transmembrane proteins on force transmission in skeletal muscle. In this study, a 2D finite element model of single muscle fiber composed of myofiber, ECM, and the transmembrane proteins between them was developed to determine how changes in spatial density and mechanical properties of transmembrane proteins affect the force transmission in skeletal muscle. We found that force transmission and stress distribution are not affected by mechanical stiffness of the transmembrane proteins due to its non-linear stress–strain relationship. Results also showed that the muscle fiber with insufficient transmembrane proteins near the end of muscle fiber transmitted less force than that with more proteins does. Higher stress was observed in myofiber, ECM, and proteins in the muscle fiber with fewer proteins.     

Age-related changes in the mechanical properties of the epimysium in skeletal muscles of rats

Skeletal muscle is composed of muscle fibers and an extracellular matrix (ECM). The collagen fiber network of the ECM is a major contributor to the passive force of skeletal muscles at high strain. We investigated the effect of aging on the biomechanical and structural properties of epimysium of the tibialis anterior muscles (TBA) of rats to understand the mechanisms responsible for the age-related changes. The biomechanical properties were tested directly in vitro by uniaxial extension of epimysium. The presence of age-related changes in the arrangement and size of the collagen fibrils in the epimysium was examined by scanning electron microscopy (SEM). A mathematical model was subsequently developed based on the structure-function relationships that predicted the compliance of the epimysium. Biomechanically, the epimysium from old rats was much stiffer than that of the young rats. No differences were found in the ultrastructure and thickness of the epimysium or size of the collagen fibrils between young and old rats. The changes in the arrangement and size of the collagen fibrils do not appear to be the principal cause of the increased stiffness of the epimysium from the old rats. Other changes in the structural composition of the epimysium from old rats likely has a strong effect on the increased stiffness. The age-related increase in the stiffness of the epimysium could play an important role in the impaired lateral force transmission in the muscles of the elderly.     

Macroscopic-microscopic characterization of the passive mechanical properties in rat soleus muscle

The purpose of the study was to investigate changes in passive mechanical properties of the soleus muscle of the rat during the first year of life. These mechanical changes were quantified at a macroscopic (whole muscle) and a microscopic level (fiber) and were correlated with biochemical and morphological properties. Three passive mechanical tests (a relaxation test, a ramp stretch test and a stretch release cycle test) with different amplitudes and velocities were performed on isolated soleus muscles and fibers in rats at ages 1 (R1), 4 (R4) and 12 (R12) months. Mechanical parameters (dynamic and static forces, stresses and normalized stiffness) were recorded and measured. The morphological properties (size of fibers and muscles) for the three groups of rats were assessed by light microscopy which allowed us to observe the evolution of the fiber type (I, IIc and IIa) in the belly region and along the longitudinal axis of the muscle. In addition, biochemical analyses were performed at the level of the whole muscle in order to determine the collagen content. The results of the passive mechanical properties between the macroscopic (muscle) and microscopic (fiber) levels showed a similar evolution. Thus, an increase of the dynamic and static forces appeared between 1 and 4 months while a decrease of the passive tension occurred between 4 and 12 months. These mechanical changes were correlated to the morphological properties. In addition, the size of the three fibers type which grew with age could explain the increase of forces between 1 and 4 months. Furthermore, the biochemical analysis showed an increase of the collagen content during the same period which could also be associated with the increase of the passive forces. After 4 months, the passive tension decreased while the size of the fiber continued to increase. The biochemical analysis showed a decrease of the collagen content after 4 months, which could explain the loss of passive tension in the whole muscle. Concerning the similar loss at the fiber level, other assumptions are required such as a myofibril loss process and an increase of intermyofibrillar spaces. The originality of this present study was to compare the passive mechanical properties between two different levels of anatomical organization within the soleus muscle of the rat and to explain these mechanical changes in terms of biochemical and morphological properties.     

Mechanical properties of the gastrocnemius aponeurosis in wild turkeys

In many muscles, the tendinous structures include both an extramuscular free tendon as well as a sheet-like aponeurosis. In both free tendons and aponeuroses the collagen fascicles are oriented primarily longitudinally, along the muscle's line of action. It is generally assumed that this axis represents the direction of loading for these structures. This assumption is well founded for free tendons, but aponeuroses undergo a more complex loading regime. Unlike free tendons, aponeuroses surround a substantial portion of the muscle belly and are therefore loaded both parallel (longitudinal) and perpendicular (transverse) to a muscle's line of action when contracting muscles bulge to maintain a constant volume. Given this biaxial loading pattern, it is critical to understand the mechanical properties of aponeuroses in both the longitudinal and transverse directions. In this study, we use uniaxial testing of isolated tissue samples from the aponeurosis of the lateral gastrocnemius of wild turkeys to determine mechanical properties of samples loaded longitudinally (along the muscle's line of action) and transversely (orthogonal to the line of action). We find that the aponeurosis has a significantly higher Young's modulus in the longitudinal than in the transverse direction. Our results also show that aponeuroses can behave as efficient springs in both the longitudinal and transverse directions, losing little energy to hysteresis. We also test the failure properties of aponeuroses to quantify the likely safety factor with which these structures operate during muscular force production. These results provide an essential foundation for understanding the mechanical function of aponeuroses as biaxially loaded biological springs.     

Passive mechanics of canine internal abdominal muscles.

The internal abdominal muscles are biaxially loaded in vivo, and therefore length-tension relations along and transverse to the directions of the muscle fibers are important in understanding their mechanical properties. We hypothesized that 1) internal oblique and transversus abdominis form an internal abdominal composite muscle with altered compliance than that of either muscle individually, and 2) anisotropy, different compliances in orthogonal directions, of internal abdominal composite muscle is less pronounced than that of its individual muscles. To test these hypotheses, in vitro mechanical testing was performed on 5 x 5 cm squares of transversus abdominis, internal oblique, and the two muscles together as a composite. These tissues were harvested from the left lateral side of abdominal muscles of eleven mongrel dogs (15-23 kg) and placed in a bath of oxygenated Krebs solution. Each tissue strip was attached to a biaxial mechanical testing device. Each muscle was passively lengthened and shortened along muscle fibers, transverse to fibers, or simultaneously along and transverse to muscle fibers. Both transversus abdominis and internal oblique muscles demonstrated less extensibility in the direction transverse to muscle fibers than along fibers. Biaxial loading caused a stiffening effect that was greater in the direction along the fibers than transverse to the fibers. Furthermore, the abdominal muscle composite was less compliant than either muscle alone in the direction of the muscle fibers. Taken together, our data suggested that the internal abdominal composite tissue has complex mechanical properties that are dependent on the mechanical properties of internal oblique and transversus abdominis muscles.     

Muscle extracellular matrix applies a transverse stress on fibers with axial strain

It is widely assumed that skeletal muscle contraction is isovolumic. This assumption has been verified at the single fiber and at the myofibril level. Model development and mechanical analyses often exploit this assumption when investigating skeletal muscle and evaluating muscle mechanical properties. This communication describes a method whereby individual muscle fibers and bundles of fibers, which include their constituent extracellular matrix (ECM), were tested to define the change in volume with axial strain. The results demonstrate that fibers are isovolumic, but bundles decrease in volume with strain. The loss of volume implicates a transverse force being applied to the fibers by the ECM. The nature and importance of this transverse force warrant further investigation.     

Muscle performance during maximal isometric and dynamic contractions is influenced by the stiffness of the tendinous structures.

Contractile force is transmitted to the skeleton through tendons and aponeuroses, and, although it is appreciated that the mechanocharacteristics of these tissues play an important role for movement performance with respect to energy storage, the association between tendon mechanical properties and the contractile muscle output during high-force movement tasks remains elusive. The purpose of the study was to investigate the relation between the mechanical properties of the connective tissue and muscle performance in maximal isometric and dynamic muscle actions. Sixteen trained men participated in the study. The mechanical properties of the vastus lateralis tendon-aponeurosis complex were assessed by ultrasonography. Maximal isometric knee extensor force and rate of torque development (RTD) were determined. Dynamic performance was assessed by maximal squat jumps and countermovement jumps on a force plate. From the vertical ground reaction force, maximal jump height, jump power, and force-/velocity-related determinants of jump performance were obtained. RTD was positively related to the stiffness of the tendinous structures (r = 0.55, P < 0.05), indicating that tendon mechanical properties may account for up to 30% of the variance in RTD. A correlation was observed between stiffness and maximal jump height in squat jumps and countermovement jumps (r = 0.64, P < 0.05 and r = 0.55, P < 0.05). Power, force, and velocity parameters obtained during the jumps were significantly correlated to tendon stiffness. These data indicate that muscle output in high-force isometric and dynamic muscle actions is positively related to the stiffness of the tendinous structures, possibly by means of a more effective force transmission from the contractile elements to the bone.     

Desmin integrates the three-dimensional mechanical properties of muscles

Striated muscle is a linear motor whose properties have been defined in terms of uniaxial structures. The question addressed here is what contribution is made to the properties of this motor by extramyofilament cytoskeletal structures that are not aligned in parallel with the myofilaments. This question arose from observations that transverse loads increase muscle force production in diaphragm but not in the hindlimb muscle, thereby indicating the presence of structures that couple longitudinal and transverse properties of diaphragmatic muscle. Furthermore, we find that the diaphragms of null mutants for the cytoskeletal protein desmin show 1) significant reductions in coupling between the longitudinal and transverse properties, indicating for the first time a role for a specific protein in integrating the three-dimensional mechanical properties of muscle, 2) significant reductions in the stiffness and viscoelasticity of muscle, and 3) significant increases in tetanic force production. Thus desmin serves a complex mechanical function in diaphragm muscle by contributing both to passive stiffness and viscoelasticity and to modulation of active force production in a three-dimensional structural network. Our finding changes the paradigm of force transmission among cells by placing our understanding of the function of the cytoskeleton in the context of the structural and mechanical complexity of muscles.     

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.     

Finite element analysis of mechanics of lateral transmission of force in single muscle fiber

Most of the myofibers in long muscles of vertebrates terminate within fascicles without reaching either end of the tendon, thus force generated in myofibers has to be transmitted laterally through the extracellular matrix (ECM) to adjacent fibers; which is defined as the lateral transmission of force in skeletal muscles. The goal of this study was to determine the mechanisms of lateral transmission of force between the myofiber and ECM. In this study, a 2D finite element model of single muscle fiber was developed to study the effects of mechanical properties of the endomysium and the tapered ends of myofiber on lateral transmission of force. Results showed that most of the force generated is transmitted near the end of the myofiber through shear to the endomysium, and the force transmitted to the end of the model increases with increased stiffness of ECM. This study also demonstrated that the tapered angle of the myofiber ends can reduce the stress concentration near the myofiber end while laterally transmitting force efficiently.     

Mast cells can modulate leukocyte accumulation and skeletal muscle function following hindlimb unloading.

Rodent hindlimb suspension is widely used to induce inflammation and muscle impairment. We set out to define the role of mast cells in neutrophil and macrophage recruitment and muscle recovery after unloading-reloading. We hypothesized that mechanical perturbation would stimulate release of proinflammatory substances by mast cells, which would influence leukocyte recruitment and muscle function. Rats were suspended for 10 days and injected with a mast cell inhibitor (cromolyn) or stimulator (compound 48/80) or a placebo before reloading. Leukocyte accumulation and muscle function were assessed using immunohistological staining and measurements of contractile properties in vitro. Our results showed that mechanical loading activated mast cells, thereby influencing leukocyte recruitment in the early reloading periods. Indeed, the inhibition of mast cell degranulation significantly reduced the number of neutrophil cell profiles in reloaded soleus muscle, whereas mast cell activation provoked a significant increase in the number of neutrophil cell profiles in uninjured muscle. However, the inhibition of mast cell degranulation also led to a significant increase in the number of ED1+ macrophage cell profiles. These perturbations in the inflammatory response caused by mast cell inhibition induced a short protective effect on the loss of muscle force after 1 day of reloading but delayed the return to the normal contractile properties of muscles after 14 days of reloading. These results indicate that mechanical loading can induce mast cell degranulation, which can influence leukocyte influx and muscle function, and also highlighted the possibility that leukocytes may play a dual role in skeletal muscles.     

Calcium-independent phospholipase A2 modulates cytosolic oxidant activity and contractile function in murine skeletal muscle cells.

Phospholipase A2 (PLA2) activity supports production of reactive oxygen species (ROS) by mammalian cells. In skeletal muscle, endogenous ROS modulate the force of muscle contraction. We tested the hypothesis that skeletal muscle cells constitutively express the calcium-independent PLA2 (iPLA2) isoform and that iPLA2 modulates both cytosolic oxidant activity and contractile function. Experiments utilized differentiated C2C12 myotubes and a panel of striated muscles isolated from adult mice. Muscle preparations were processed for measurement of mRNA by real-time PCR, protein by immunoblot, cytosolic oxidant activity by the dichlorofluorescein oxidation assay, and contractile function by in vitro testing. We found that iPLA2 was constitutively expressed by all muscles tested (myotubes, diaphragm, soleus, extensor digitorum longus, gastrocnemius, heart) and that mRNA and protein levels were generally similar among muscles. Selective iPLA2 blockade by use of bromoenol lactone (10 microM) decreased cytosolic oxidant activity in myotubes and intact soleus muscle fibers. iPLA2 blockade also inhibited contractile function of unfatigued soleus muscles, shifting the force-frequency relationship rightward and depressing force production during acute fatigue. Each of these changes could be reproduced by selective depletion of superoxide anions using superoxide dismutase (1 kU/ml). These findings suggest that constitutively expressed iPLA2 modulates oxidant activity in skeletal muscle fibers by supporting ROS production, thereby influencing contractile properties and fatigue characteristics.