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Abstract: We used a gait analysis system (GAS™) to measure the changes in locomotion parameters of adult Sprague–Dawley rats after neuromuscular injury, induced by repeated large-strain lengthening contractions of the dorsiflexors muscles. We developed a logistic regression model from test runs of control and permanently impaired (denervation of the dorsiflexor muscles) rats and used this model to predict the probabilities of locomotory impairment in rats injured by lengthening contractions. The data showed that GAS™ predicts the probability of locomotory impairment with very high reliability, with values close to 100% immediately after injury and close to 0% after several weeks of recovery from injury. The six transformed locomotion parameters most effective in the model were in three domains: frequency, force, and time. We conclude that application of the GAS™ instrument with our predictive model accurately identifies locomotory changes due to neuromuscular deficits. Use of this technology should be valuable for monitoring the progression of a neuromuscular disease and the effects of therapeutic interventions. [Copyright &y& Elsevier]

Abstract: Lovering RM, Roche JA, Bloch RJ, De Deyne PG. Recovery of function in skeletal muscle following 2 different contraction-induced injuries. Objective: To determine if the proliferation of myogenic cells is equally important to recovery of contractile function after 2 different types of contraction-induced muscle injuries. Design: Randomized trial. Setting: Muscle biology laboratory. Animals: Adult male Sprague-Dawley rats. Interventions: Tibialis anterior muscles were injured by a single lengthening contraction with large strain (1R) or multiple lengthening contractions with small strain (MR). The hindlimbs of some animals in each group were irradiated before injury to prevent proliferation of myogenic cells during recovery. Main Outcome Measures: Contractile tension was measured immediately after injury and 3, 7, 14, and 21 days after injury. Permeation to Evans blue dye was used to assay membrane damage. Centrally nucleated fibers and reverse transcriptase-polymerase chain reaction of myoD and myogenin were used as measures of myogenesis. Results: Inhibiting myogenesis prevented the recovery of contractile function after MR, but not after 1R. Both protocols caused Evans blue dye uptake immediately after injury, but Evans blue dye was only retained in fibers for several days after 1R. This suggests that membranes reseal after 1R, but not after MR. Conclusions: The mechanisms that underlie recovery after injuries caused by repeated lengthening contractions and injuries caused by a single lengthening contraction are different. The differences may be important when planning targeted rehabilitation strategies for each type of injury. [Copyright &y& Elsevier]

Abstract: Purpose: Some muscle injuries are the result of a single lengthening contraction. Our goal was to evaluate the contributions of angular velocity, arc of motion, and timing of contractile activation relative to the onset of joint motion in an animal model of muscle injury using a single lengthening contraction. Methods: The intact tibialis anterior (TA) muscle of rats was activated while lengthened, preceded by a maximal isometric contraction of 0, 25, 50, 100, or 200ms. The lengthening contraction was performed at two different angular velocities (300 or 900°/s) and through two different arcs of motion (90° or 45°). Results: Muscle contractile function, as measured by maximal isometric tetanic tension, was significantly decreased only when the TA was activated at least 50ms prior to the motion, regardless of angular velocity or arc of motion. Conclusion: The data indicated that the duration of an isometric contraction prior to a single lengthening contraction determined the extent of muscle injury irrespective of two different angular velocities. [Copyright &y& Elsevier]

• Duchenne muscular dystrophy (DMD), the most common form of muscular dystrophy, is caused by the lack of dystrophin, a protein encoded by the DMD gene. • The neuromuscular junction (NMJ) is not a fixed, permanent structure, but instead shows plasticity in response to injury, exercise, and aging. • NMJs in the mdx mouse model of DMD show aberrant changes in both pre- and post-synaptic NMJ structure, which could influence neuromuscular transmission. • The hypothesis that the NMJ contributes to functional deficits in DMD represents a paradigm shift from more prevalent myo-centric perspectives. The focus of this review is on Duchenne muscular dystrophy (DMD), which is caused by the absence of the protein dystrophin and is characterized as a neuromuscular disease in which muscle weakness, increased susceptibility to muscle injury, and inadequate repair appear to underlie the pathology. Considerable attention has been dedicated to studying muscle fiber damage, but data show that both human patients and animal models for DMD present with fragmented neuromuscular junction (NMJ) morphology. In addition to pre- and post-synaptic abnormalities, studies indicate increased susceptibility of the NMJ to contraction-induced injury, with corresponding functional changes in neuromuscular transmission and nerve-evoked electromyographic activity. Such findings suggest that alterations in the NMJ of dystrophic muscle may play a role in muscle weakness via impairment of neuromuscular transmission. Further work is needed to fully understand the role of the NMJ in the weakness, susceptibility to injury, and progressive wasting associated with DMD. [ABSTRACT FROM AUTHOR]

Cells respond to their mechanical environment by initiating multiple mechanotransduction signaling pathways. Defects in mechanotransduction have been implicated in a number of pathologies; thus, there is need for convenient and efficient methods for studying the mechanisms underlying these processes. A widely used and accepted technique for mechanically stimulating cells in culture is the introduction of fluid flow on cell monolayers. Here, we describe a novel, multifunctional fluid flow device for exposing cells to fluid flow in culture. This device integrates with common lab equipment including routine cell culture plates and peristaltic pumps. Further, it allows the fluid flow treated cells to be examined with outcomes at the cell and molecular level. We validated the device using the biologic response of cultured UMR-106 osteoblast-like cells in comparison to a commercially available system of laminar sheer stress to track live cell calcium influx in response to fluid flow. In addition, we demonstrate the fluid flow-dependent activation of phospho-ERK in these cells, consistent with the findings in other fluid flow devices. This device provides a low cost, multi-functional alternative to currently available systems, while still providing the ability to generate physiologically relevant conditions for studying processes involved in mechanotransduction in vitro . [ABSTRACT FROM AUTHOR]

Tissue engineering strategies to treat patients with volumetric muscle loss (VML) aim to recover the structure and contractile function of lost muscle tissue. Here, we assessed the capacity of novel electrospun fibrin hydrogel scaffolds seeded with murine myoblasts to regenerate the structure and function of damaged muscle within VML defects to the mouse tibialis anterior muscle. The electrospun fibrin scaffolds provide pro-myogenic alignment and stiffness cues, myomimetic hierarchical structure, suturability, and scale-up capabilities. Myoblast-seeded scaffolds enabled remarkable muscle regeneration with high myofiber and vascular densities after 2 and 4 weeks, mimicking that of native skeletal muscle, while acellular scaffolds lacked muscle regeneration. Both myoblast-seeded and acellular scaffolds fully recovered muscle contractile function to uninjured values after 2 and 4 weeks. Electrospun scaffolds pre-vascularized with co-cultured human endothelial cells and human adipose-derived stem cells implanted into VML defects for 2 weeks anastomosed with host vasculature and were perfused with host red blood cells. These data demonstrate the significant potential of electrospun fibrin scaffolds seeded with myoblasts to fully regenerate the structure and function of volumetric muscle defects and these scaffolds offer a promising treatment option for patients with VML. [ABSTRACT FROM AUTHOR]

Abstract: Most animal studies of muscle contractile function utilise the anterior or posterior crural muscle (dorsiflexors and plantarflexors, respectively). An advantage to using these muscles is that the common fibular and tibial nerves are readily accessible, while the small size of the crural muscles is a disadvantage. Working with small muscles not only makes some in vivo imaging and the muscle testing techniques more challenging, but also provides limited amounts of tissue to study. The purpose of this study was to describe a new animal muscle injury model in the quadriceps that results in a significant and reproducible loss of force. The thigh of Sprague Dawley rats (N =5) and C57BL/10 mice (N =5) was immobilised and the ankle was attached to a custom-made lever arm. The femoral nerve was stimulated using subcutaneous electrodes and injury was induced using 50 lengthening (“eccentric”) contractions through a 70° arc of knee motion. This protocol produces a significant and reproducible injury, with comparable susceptibility to injury in the rats and mice. This novel model shows that the quadriceps muscle provides a means to study whole muscle contractility, injury, and recovery in vivo. In addition to the usual benefits of an in vivo model, the larger size of the quadriceps facilitates in vivo imaging and provides a significant increase in the amount of tissue available for histology and biochemistry studies. A controlled muscle injury in the quadriceps also allows one to study a muscle, with mixed fibre types, which is extremely relevant to gait in humans and quadruped models. [Copyright &y& Elsevier]

Abstract: The intermediate filament-like protein syncoilin is a member of the dystrophin protein complex, and links the complex to the cytoskeleton through binding α-dystrobrevin and desmin in muscle. Here, we identify further sites of syncoilin location in normal muscle: at the perinuclear space, myotendinous junction, and enrichment in the sarcolemma and sarcoplasm of oxidative muscle fibers in mice. To understand the importance of the dystrophin protein complex-syncoilin-cytoskeletal link and its implication to disease, we analyzed syncoilin in mice null for α-dystrobrevin (adbn−/−) and desmin (des−/−). Syncoilin was upregulated in dystrophic muscles of adbn−/− mice, without alteration in its subcellular location. In des−/− mice, syncoilin was severely reduced in skeletal muscle; lost from sarcomeric Z-lines and neuromuscular junctions, and redistributed from the sub-sarcolemmal cytoskeleton to the cytoplasm. The data show that absence of α-dystrobrevin or desmin leads to dynamic changes in syncoilin that may compensate for, or participate in, different muscle myopathies. [Copyright &y& Elsevier]

Mechanical forces are conducted through myofibers and into nuclei to regulate muscle development, hypertrophy, and homeostasis. We hypothesized that nuclei in aged muscle have changes in the nuclear envelope and associated proteins, resulting in altered markers of mechano-signaling. YAP/TAZ protein expression and gene expression of downstream targets, Ankrd1 and Cyr61 , were evaluated as mechanotransduction indicators. Expression of proteins in the nuclear lamina and the nuclear pore complex (NPC) were assessed, and nuclear morphology was characterized by electron microscopy. Nuclear envelope permeability was assessed by uptake of 70 kDa fluorescent dextran. Nuclear changes with aging included a relative decrease of lamin β1 and Nup107, and a relative increase in Nup93, which could underlie the aberrant nuclear morphology, increased nuclear leakiness, and elevated YAP/TAZ signaling. Aged muscles have hyperactive nuclear-cytoplasmic signaling, indicative of altered nuclear mechanotransduction. These data highlight a possible role for the nucleus in aging-related aberrant mechano-sensing. [ABSTRACT FROM AUTHOR]

Volumetric muscle loss (VML) overwhelms the native regenerative capabilities of skeletal muscle and has few effective treatments to regain lost muscle mass and function. Tissue engineered muscle constructs designed to promote neuromuscular regeneration are a promising therapeutic avenue. To date, there has been no engineered muscle construct for VML treatment that has incorporated a pharmacologic agent to promote neuromuscular regeneration. Here, we have modified electrospun fibrin microfiber bundles, which have demonstrated muscle regenerative potential, with the heparan sulfate proteoglycan, agrin, to stimulate innervation post-VML. Myoblasts cultured on microfiber bundles with either soluble or chemically tethered agrin demonstrated statistically significant increased clustering of acetylcholine receptors (AChRs) with soluble agrin displaying AChR clusters throughout the myofiber bundles, and tethered agrin displaying AChR clusters only at 10 μm from the substrate surface. Following implantation into murine VML defects for 4 weeks, constructs pre-treated with soluble or tethered agrin resulted in statistically significant increased neuromuscular junctions, regenerating myofibers, vascular infiltration, neural infiltration, and nuclear yes-associated protein (YAP) expression within the defect site compared to the control without agrin. The agrin-tethered microfiber bundles provided sustained agrin signaling within the regenerating site during the 4-week post-implantation periods and further augmented the density of regenerating myofibers in regenerated tissue with statistical significance compared to constructs with soluble agrin. These data demonstrate the neuromuscular regenerative potential of engineered muscle constructs pre-treated to induce AChR clustering with locally delivered agrin at the site of VML regeneration. [ABSTRACT FROM AUTHOR]