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ABSTRACT Introduction: We examined the possibility that tetanus toxin can prevent muscle atrophy associated with limb immobility in rats. Methods: While the knee and ankle joints were immobilized unilaterally, the tibialis anterior (TA) muscle on the immobilized side was injected with 1 μl saline or with 1 ng tetanus toxin. After 2 weeks, TA wet weights, contractile forces, and myofiber sizes from the immobilized sides were compared with those from body weight-matched normal animals. Results: Saline group wet weights decreased and produced less absolute twitch and tetanic force and normalized tetanic force compared with the toxin or normal groups. Cross-sectional areas of saline group type I, IIa, and IId myofibers, and the masses of saline group IIa, IId, IIb, and toxin group IIb myofibers, were smaller compared with the normal group. Conclusions: Tetanus toxin prevented common signs of muscle atrophy and may become a useful adjunct to current rehabilitation strategies. Muscle Nerve 50: 759-766, 2014 [ABSTRACT FROM AUTHOR]

Neurotrophin-dependent activation of the tyrosine kinase receptor trkB.FL modulates neuromuscular synapse maintenance and function; however, it is unclear what role the alternative splice variant, truncated trkB (trkB.T1), may have in the peripheral neuromuscular axis. We examined this question in trkB.T1 null mice and demonstrate that in vivo neuromuscular performance and nerve-evoked muscle tension are significantly increased. In vitro assays indicated that the gain-in-function in trkB.T1-/- animals resulted specifically from an increased muscle contractility, and increased electrically evoked calcium release. In the trkB.T1 null muscle, we identified an increase in Akt activation in resting muscle as well as a significant increase in trkB.FL and Akt activation in response to contractile activity. On the basis of these findings, we conclude that the trkB signaling pathway might represent a novel target for intervention across diseases characterized by deficits in neuromuscular function. [ABSTRACT FROM AUTHOR]

Intermediate filaments, composed of desmin and of keratins, play important roles in linking contractile elements to each other and to the sarcolemma in striated muscle. Our previous results show that the tibialis anterior (TA) muscles of mice lacking keratin 19 (K19) lose costameres, accumulate mitochondria under the sarcolemma, and generate lower specific tension than controls. Here we compare the physiology and morphology of TA muscles of mice lacking K19 with muscles lacking desmin or both proteins [double knockout (DKO)]. K19-/- mice and DKO mice showed a threefold increase in the levels of creatine kinase (CK) in the serum. The absence of desmin caused a larger change in specific tension (-40%) than the absence of K19 (-19%) and played the predominant role in contractile function (-40%) and decreased tolerance to exercise in the DKO muscle. By contrast, the absence of both proteins was required to obtain a significantly greater loss of contractile torque after injury (-48%) compared with wild type (-39%), as well as near-complete disruption of costameres. The DKO muscle also showed a significantly greater misalignment of myofibrils than either mutant alone. In contrast, large subsarcolemmal gaps and extensive accumulation of mitochondria were only seen in K19-null TA muscles, and the absence of both K19 and desmin yielded milder phenotypes. Our results suggest that keratin filaments containing K19- and desmin-based intermediate filaments can play independent, complementary, or antagonistic roles in the physiology and morphology of fast-twitch skeletal muscle. [ABSTRACT FROM AUTHOR]

Abstract: Insulin-like growth factor (IGF-I) is hypothesized to be a critical upstream regulator of mammalian target of rapamycin (mTOR)-regulated protein synthesis with muscle contraction. We utilized a mouse model that expresses a skeletal muscle specific dominant-negative IGF-I receptor to investigate the role of IGF-I signaling of protein synthesis in response to unilateral lengthening contractions (10 sets, 6 repetitions, 100Hz) at 0 and 3h following the stimulus. Our results indicate that one session of high frequency muscle contractions can activate mTOR signaling independent of signaling components directly downstream of the receptor. [Copyright &y& Elsevier]

We studied the response of dysferlin-null and control skeletal muscle to largeand small-strain injuries to the ankle dorsiflexors in mice. We measured contractile torque and counted fibers retaining 10-kDa fluorescein dextran, necrotic fibers, macrophages, and fibers with central nuclei and expressing developmental myosin heavy chain to assess contractile function, membrane resealing, necrosis, inflammation, and myogenesis. We also studied recovery after blunting myogenesis with X-irradiation. We report that dysferlin-null myofibers retain 10-kDa dextran for 3 days after large-strain injury but are lost thereafter, following necrosis and inflammation. Recovery of dysferlin-null muscle requires myogenesis, which delays the return of contractile function compared with controls, which recover from large-strain injury by repairing damaged myofibers without significant inflammation, necrosis, or myogenesis. Recovery of control and dysferlin-null muscles from small-strain injury involved inflammation and necrosis followed by myogenesis, all of which were more pronounced in the dysferlin-null muscles, which recovered more slowly. Both control and dysferlin-null muscles also retained 10-Wa dextran for 3 days after small-strain injury. We conclude that dysferlin-null myofibers can survive contraction-induced injury for at least 3 days but are subsequently eliminated by necrosis and inflammation. Myogenesis to replace lost fibers does not appear to be significantly compromised in dysferlin-null mice. [ABSTRACT FROM AUTHOR]

High-force lengthening contractions are associated with muscle damage and pain, and the muscle-tendon junction is commonly cited as the primary area where myofiber damage occurs. We induced injury in the rat tibialis anterior muscle and acquired magnetic resonance imaging (MRI) images postinjury. We also assayed membrane damage and quantified the number of centrally nucleated myofibers throughout the injured muscles. Results suggest that myofiber injury occurs primarily in the middle portion of the muscle, with interstitial edema in the middle and distal portions. Muscle Nerve, 2009 [ABSTRACT FROM AUTHOR]

Skeletal muscle function is dependent on its highly regular structure. In studies of dystrophic (dy/dy) mice, the proportion of malformed myofibers decreases after prolonged whole muscle stimulation, suggesting that the malformed myofibers are more prone to injury. The aim of this study was to assess morphology and to measure excitation-contraction (EC) coupling (Ca2+ transients) and susceptibility to osmotic stress (Ca2+ sparks) of enzymatically isolated muscle fibers of the extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles from young (2-3 mo) and old (8-9 mo) mdx and age-matched control mice (C57BLIO). In young mdx EDL, 6% of the myofibers had visible malformations (i.e., interfiber splitting, branched ends, midfiber appendages). In contrast, 65% of myofibers in old mdx EDL contained visible malformations. In the mdx FDB, malformation occurred in only 5% of young myofibers and 11% of old myofibers. Age-matched control mice did not display the altered morphology of mdx muscles. The membrane-associated and cytoplasmic cytoskeletal structures appeared normal in the malformed mdx myofibers. In mdx FDBs with significantly branched ends, an assessment of global, electrically evoked Ca2+ signals (indo-IPE-AM) revealed an EC coupling deficit in myofibers with significant branching. Interestingly, peak amplitude of electrically evoked Ca2+ release in the branch of the bifurcated mdx myofiber was significantly decreased compared with the trunk of the same myofiber. No alteration in the basal myoplasmic Ca2+ concentration (i.e., indo ratio) was seen in malformed vs. normal mdx myofibers. Finally, osmotic stress induced the occurrence of Ca2+ sparks to a greater extent in the malformed portions of myofibers, which is consistent with deficits in EC coupling control. In summary, our data show that aging mdx myofibers develop morphological malformations. These malformations are not associated with gross disruptions in cytoskeletal or t-tubule structure; however, alterations in myofiber Ca2+ signaling are evident. [ABSTRACT FROM AUTHOR]

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]

The paired pyramidalis muscles are small triangular-shaped muscles that lie between the anterior surface of the rectus abdominus and the posterior surface of the rectus sheath. The precise function of pyramidalis muscles is unclear, but together the muscles are thought to tense the linea alba. The muscles are not always present, or are often unilateral, and vary greatly in size. Their wider inferior margins attach to the pubic symphyses and pubic crests, whereas their narrow superior margins attach to the linea alba. The gross anatomy and innervation of the pyramidalis muscles has been described by others, but their architecture and fiber type have not been determined in previous publications. The purpose of the present paper was therefore to investigate these parameters and place the findings into context for the literature available on this muscle. An example of bilateral pyramidalis muscles was recently encountered in a male cadaver that provided ample tissue for an analysis of its architecture and fiber type. The muscle mass, muscle length, fiber length, and pennation angle of muscle fibers were measured to ascertain physiological cross-sectional area and thereby estimate force production. Fiber type composition was also examined using immunofluorescent labeling. The results show that this is a muscle of mixed fiber type composition, similar to the rectus abdominus, and that the estimated forces generated by this muscle are relatively small. [ABSTRACT FROM AUTHOR]

The genetic basis of many muscular disorders, including many of the more common muscular dystrophies, is now known. Clinically, the recent genetic advances have improved diagnostic capabilities, but they have not yet provided clues about treatment or management. Thanks to better management strategies and therapeutic interventions, however, many patients with a muscular dystrophy are more active and are living longer. Physical therapists, therefore, are more likely to see a patient with a muscular dystrophy, so understanding these muscle disorders and their management is essential. Physical therapy offers the most promise in caring for the majority of patients with these conditions, because it is unlikely that advances in gene therapy will significantly alter their clinical treatment in the near future. This perspective covers some of the basic molecular biological advances together with the clinical manifestations of the muscular dystrophies and the latest approaches to their management. [ABSTRACT FROM AUTHOR]

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]

Injuries to the anterior cruciate ligament (ACL) result in immediate and long-term morbidity and expense. Young women are more likely to sustain ACL injuries than men who participate in similar athletic and military activities. Although significant attention has focused on the role that female sex hormones may play in this disparity, it is still unclear whether the female ACL also responds to androgens. The purpose of this study was to determine whether the female ACL was an androgen-responsive tissue. To identify and localize androgen receptors in the female ACL, we used Western blotting and immunofluorescent labeling, respectively, of ACL tissue harvested during surgery from young women (n = 3). We then measured ACL stiffness and assessed total testosterone (T) and free [free androgen index (FAI)] testosterone concentrations, as well as relative estradiol to testosterone ratios (E2/T and E2/FAI) at three consecutive menstrual stages (n = 20). There were significant rank-order correlations between T (0.48, P = 0.031), FAI (0.44, P = 0.033), E2/T (-0.71, P < 0.001), E2/FAI (-0.63, P = 0.003), and ACL stiffness near ovulation. With the influences of the other variables controlled, them were significant negative partial rank-order correlations between ACL stiffness and E2/T (-0.72, P < 0.001) and E2/FAI (-0.59, P = 0.012). The partial order residuals for T and FAI were not significant. These findings suggest that the female ACL is an androgen-responsive tissue but that T and FAI are not independent predictors of ACL stiffness near ovulation. Instead, the relationship between T, FAI, and ACL stiffness was likely influenced by another hormone or sex hormone binding globulin. [ABSTRACT FROM AUTHOR]

The purpose of this study was to evaluate the integrity of the muscle membrane and its associated cytoskeleton after a contraction-induced injury. A single eccentric contraction was performed in vivo on the tibialis anterior (TA) of male SpragueDawley rats at 900°/s throughout a 90°-arc of motion. Maximal tetanic tension (Po) of the TAs was assessed immediately and at 3, 7, and 21 days after the injury. To evaluate sarcolemmal integrity, we used an Evans blue dye (EBD) assay, and to assess structural changes, we used immunofluorescent labeling with antibodies against contractile (myosin, actin), cytoskeletal (α-actinin, desmin, dystrophin, β-spectrin), integral membrane (α- and β-dystroglycan, sarcoglycan), and extracellular (laminin, fibronectin) proteins. Immediately after injury, Po was significantly reduced to 4.23 ± 0.22 N, compared with 8.24 ± 1.34 N in noninjured controls, and EBD was detected intracellularly in 54 ± 22% of fibers from the injured TA, compared with 0% in noninjured controls. We found a significant association between EBD-positive fibers and the loss of complete dystrophin labeling. The loss of dystrophin was notable because organization of other components of the subsarcolemmal cytoskeleton was affected minimally (β-spectrin) or not at all (α- and β-dystroglycan). Labeling with specific antibodies indicated that dystrophin's COOH terminus was selectively more affected than its rod domain. Twenty-one days after injury, contractile properties were normal, fibers did not contain EBD, and dystrophin organization and protein level returned to normal. These data indicate the selective vulnerability of dystrophin after a single eccentric contraction-induced injury and suggest a critical role of dystrophin in force transduction. muscle injury; dystrophin; cytoskeleton; sarcolemma. [ABSTRACT FROM AUTHOR]

• 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]

Introduction: The ability to view individual myofibers is possible with many histological techniques, but not yet with standard in vivo imaging. Optical coherence tomography (OCT) is an emerging technology that can generate high resolution 1-10 μm cross-sectional imaging of tissue in vivo and in real time. Methods: We used OCT to determine architectural differences of tibialis anterior muscles in situ from healthy mice (wild-type [WT], n = 4) and dystrophic mice ( mdx, n = 4). After diffusion tensor imaging (DTI) and OCT, muscles were harvested, snap-frozen, and sectioned for staining with wheat germ agglutinin. Results: DTI suggested differences in pennation and OCT was used to confirm this supposition. OCT indicated a shorter intramuscular tendon (WT/ mdx ratio of 1.2) and an 18% higher degree of pennation in mdx. Staining confirmed these architectural changes. Conclusions: Architectural changes in mdx muscles, which could contribute to reduction of force, are detectable with OCT. Muscle Nerve, 2013 [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]

Fibroadipogenic progenitor (FAP) cells are implicated as a major source of fatty infiltration (FI) in murine rotator cuff (RC) injury, but FAP cell response after RC tear in a rabbit model is unknown. This study determined whether changes in FAP cell count after an RC tear predate muscle degeneration in a clinically relevant rabbit model. We hypothesized increases in FAP cell count correlate temporally with RC degeneration. New Zealand white rabbits (n = 26) were evaluated at 1, 2, 4, and 6 weeks after unilateral full‐thickness tenotomy of supraspinatus and infraspinatus tendons. FI area and adipocyte size were histologically analyzed, muscle density was measured by computerized tomography, and quantification of FAP cells was measured by flow cytometry and immunohistochemistry. The percentage of intrafascicular adipocyte area increased over time in supraspinatus muscle samples (p = 0.03), significantly between 1‐ and 6‐week samples (p = 0.04). There were no differences in perifascicular adipocyte area percentages between time points. Peak increase in FAP cell count occurred at 1‐week (p = 0.03), with a decrease in the following weeks. There was a negative correlation between supraspinatus adipocyte area and FAP cell count (p < 0.05). On computed tomography (CT) scan, maximal decrease in muscle density was observed in the 4th to 6th weeks. In summary, FAP cell response occurred early after tenotomy and did not correlate temporally with increases in FI. This suggests that FAP cell response may predate degenerative changes, and early targeting of FAPs before adipocyte maturation could blunt FI after RC tear. [ABSTRACT FROM AUTHOR]

Disruptions of ovarian function in women are associated with increased risk of metabolic disease due to dysregulation of peripheral glucose homeostasis in skeletal muscle. Our previous evidence suggests that alterations in skeletal muscle lipid metabolism coupled with altered mitochondrial function may also develop. The objective of this study was to use an integrative metabolic approach to identify potential areas of dysfunction that develop in skeletal muscle from ovariectomized (OVX) female mice compared with age-matched ovary-intact adult female mice (sham). The OVX mice exhibited significant increases in body weight, visceral, and inguinal fat mass compared with sham mice. OVX mice also had significant increases in skeletal muscle intramyocellular lipids (IMCL) compared with the sham animals, which corresponded to significant increases in the protein content of the fatty acid transporters CD36/FAT and FABPpm. A targeted metabolic profiling approach identified significantly lower levels of specific acyl carnitine species and various amino acids in skeletal muscle from OVX mice compared with the sham animals, suggesting a potential dysfunction in lipid and amino acid metabolism, respectively. Basal and maximal mitochondrial oxygen consumption rates were significantly impaired in skeletal muscle fibers from OVX mice compared with sham animals. Collectively, these data indicate that loss of ovarian function results in increased IMCL storage that is coupled with alterations in mitochondrial function and changes in the skeletal muscle metabolic profile. [ABSTRACT FROM AUTHOR]

The article presents a study focusing on the determinants of the repeated-bout effect (RBE) after lengthening contractions. As stated, the study compared the protective effect of two different injury protocols on the ankle dorsiflexors in the rat. The study concludes that RBE is both rapid in onset and prolonged, and not all injuries can protect against different types of subsequent injury.

The role of S100A1 in skeletal muscle is just beginning to be elucidated. We have previously shown that skeletal muscle fibers from S100A1 knockout (KO) mice exhibit decreased action potential (AP)-evoked Ca2+ transients, and that S100A1 binds competitively with calmodulin to a canonical S100 binding sequence within the calmodulinbinding domain of the skeletal muscle ryanodine receptor. Using voltage clamped fibers, we found that Ca2+ release was suppressed at all test membrane potentials in S100A1-/- fibers. Here we examine the role of S100A1 during physiological AP-induced muscle activity, using an integrative approach spanning AP propagation to muscle force production. With the voltage-sensitive indicator di-8-aminonaphthylethenylpyridinium, we first demonstrate that the AP waveform is not altered in flexor digitorum brevis muscle fibers isolated from S100A1 KO mice. We then use a model for myoplasmic Ca2+ binding and transport processes to calculate sarcoplasmic reticulum Ca2+ release flux initiated by APs and demonstrate decreased release flux and greater inactivation of flux in KO fibers. Using in vivo stimulation of tibialis anterior muscles in anesthetized mice, we show that the maximal isometric force response to twitch and tetanic stimulation is decreased in S100A1-/- muscles. KO muscles also fatigue more rapidly upon repetitive stimulation than those of wild-type counterparts. We additionally show that fiber diameter, type, and expression of key excitation-contraction coupling proteins are unchanged in S100A1 KO muscle. We conclude that the absence of S100A1 suppresses physiological AP-induced Ca2+ release flux, resulting in impaired contractile activation and force production in skeletal muscle. [ABSTRACT FROM AUTHOR]

Abstract: Bex1 and Calmodulin (CaM) are upregulated during skeletal muscle regeneration. We confirm this finding and demonstrate the novel finding that they interact in a calcium-dependent manner. To study the role of Bex1 and its interaction with CaM in skeletal muscle regeneration, we generated Bex1 knock out (Bex1-KO) mice. These mice appeared to develop normally and are fertile, but displayed a functional deficit in exercise performance compared to wild type (WT) mice. After intramuscular injection of cardiotoxin, which causes extensive and reproducible myotrauma followed by recovery, regenerating muscles of Bex1-KO mice exhibited elevated and prolonged cell proliferation, as well as delayed cell differentiation, compared to WT mice. Thus, our results provide the first evidence that Bex1-KO mice show altered muscle regeneration, and allow us to propose that the interaction of Bex1 with Ca2+/CaM may be involved in skeletal muscle regeneration. [Copyright &y& Elsevier]

The neuromuscular junction (NMJ) is a specialized synapse that bridges the motor neuron and the skeletal muscle fiber and is crucial for conversion of electrical impulses originating in the motor neuron to action potentials in the muscle fiber. The consideration of contributing factors to skeletal muscle injury, muscular dystrophy and sarcopenia cannot be restricted only to processes intrinsic to the muscle, as data show that these conditions incur denervation-like findings, such as fragmented NMJ morphology and corresponding functional changes in neuromuscular transmission. Primary defects in the NMJ also influence functional loss in motor neuron disease, congenital myasthenic syndromes and myasthenia gravis, resulting in skeletal muscle weakness and heightened fatigue. Such findings underscore the role that the NMJ plays in neuromuscular performance. Regardless of cause or effect, functional denervation is now an accepted consequence of sarcopenia and muscle disease. In this short review, we provide an overview of the pathologic etiology, symptoms, and therapeutic strategies related to the NMJ. In particular, we examine the role of the NMJ as a disease modifier and a potential therapeutic target in neuromuscular injury and disease. [ABSTRACT FROM AUTHOR]

Introduction: Our aim was to assess key muscle imaging and contractility parameters in the Duchenne muscular dystrophy (DMD) rat model (Dmd-KO rat), which have not yet been characterized sufficiently.Methods: We performed in-vivo magnetic resonance imaging (MRI) for thigh and leg muscles, and performed hematoxylin and eosin (H&E) staining and in-vivo muscle contractility testing in specific hindlimb muscles.Results: MRI prior to testing muscle contractility revealed multiple, unevenly distributed focal hyperintensities in the Dmd-KO rat quadriceps and tibialis anterior muscles. H&E staining showed corresponding areas of inflammation and ongoing regeneration. In-vivo contractile testing showed maximal force generated by Dmd-KO muscles was significantly lower, and susceptibility to injury was ~ two-fold greater in the Dmd-KO rats compared to wild-type (WT) rats.Discussion: Together, the MRI findings, histological findings, and the low strength and high susceptibility to injury in muscles support use of the Dmd-KO rat as an animal model of DMD. [ABSTRACT FROM AUTHOR]

In order to repair chronic nerve injuries (injuries repaired after a long delay), the damaged nerve segments are resected and stumps are bridged by grafts. Autografts remain the gold-standard, but outcomes are typically poor, even after long periods of recovery. In a recent study, we described the use of a nerve lengthening device to gradually elongate the proximal stump of a transected nerve towards the distal stump, enabling a tension-free end-to-end repair. This approach showed significantly improved outcomes in comparison to autografts in repairing acutely injured nerves. In this study, we compared the use of nerve lengthening/end-to-end repair (LETER) to isograft repair of chronically transected nerves in a rat model. Structural and functional regenerative outcomes following LETER were comparable to isograft-based repair, with no significant differences found in outcomes involving functional recovery or axon growth. These data demonstrate the feasibility of nerve lengthening as a viable graft-free strategy for repairing chronically injured nerves. Not unexpectedly, outcomes for chronic nerve injuries were less favorable in both groups compared to repair of acutely injured nerves. Nonetheless, the findings provide insight into barriers to restoring function after chronic nerve injury through novel comprehensive characterization of a diverse set of neuromuscular outcomes. This analysis revealed key parameters predicting functional recovery. • Recovery outcomes were similar when comparing nerve repairs by graft or lengthening. • Nerve lengthening after chronic injury is viable and avoids need to harvest graft. • Chronic and acute nerve injuries have very different outcomes. [ABSTRACT FROM AUTHOR]

Background: Clinical use of platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) has gained momentum as treatment for muscle injuries. Exosomes, or small cell–derived vesicles, could be helpful if they could deliver the same or better physiological effect without cell transplantation into the muscle. Hypothesis: Local delivery of exosomes derived from PRP (PRP-exos) or MSCs (MSC-exos) to injured muscles hastens recovery of contractile function. Study Design: Controlled laboratory study. Methods: In a rat model, platelets were isolated from blood, and MSCs were isolated from bone marrow and expanded in culture; exosomes from both were isolated through ultracentrifugation. The tibialis anterior muscles were injured in vivo using maximal lengthening contractions. Muscles were injected with PRP-exos or MSC-exos (immediately after injury and 5 and 10 days after injury); controls received an equal volume of saline. Histological and biochemical analysis was performed on tissues for all groups. Results: Injury resulted in a significant loss of maximal isometric torque (66% ± 3%) that gradually recovered over 2 weeks. Both PRP-exos and MSC-exos accelerated recovery, with similar faster recovery of contractile function over the saline-treated group at 5, 10, and 15 days after injury (P <.001). A significant increase in centrally nucleated fibers was seen with both types of exosome groups by day 15 (P <.01). Genes involved in skeletal muscle regeneration were modulated by different exosomes. Muscles treated with PRP-exos had increased expression of Myogenin gene (P <.05), whereas muscles treated with MSC-exos had reduced expression of TGF-β (P <.05) at 10 days after muscle injury. Conclusion: Exosomes derived from PRP or MSCs can facilitate recovery after a muscle strain injury in a small-animal model likely because of factors that can modulate inflammation, fibrosis, and myogenesis. Clinical Relevance: Given their small size, low immunogenicity, and ease with which they can be obtained, exosomes could represent a novel therapy for many orthopaedic ailments. [ABSTRACT FROM AUTHOR]

Intermediate filaments (IFs) contribute to force transmission, cellular integrity, and signaling in skeletal muscle. We previously identified keratin 19 (Krt19) as a muscle IF protein. We now report the presence of a second type I muscle keratin, Krt18. Krt18 mRNA levels are about half those for Krt19 and only 1:1,000th those for desmin; the protein was nevertheless detectable in immunoblots. Muscle function, measured by maximal isometric force in vivo, was moderately compromised in Krt18-knockout (Krt18-KO) or dominant-negative mutant mice (Krt18 DN), but structure was unaltered. Exogenous Krt18, introduced by electroporation, was localized in a reticulum around the contractile apparatus in wild-type muscle and to a lesser extent in muscle lacking Krt19 or desmin or both proteins. Exogenous Krt19, which was either reticular or aggregated in controls, became reticular more frequently in Krt19-null than in Krt18-null, desmin-null, or double-null muscles. Desmin was assembled into the reticulum normally in all genotypes. Notably, all three IF proteins appeared in overlapping reticular structures. We assessed the effect of Krt18 on susceptibility to injury in vivo by electroporating siRNA into tibialis anterior (TA) muscles of control and Krt19-KO mice and testing 2 wk later. Results showed a 33% strength deficit (reduction in maximal torque after injury) compared with siRNA-treated controls. Conversely, electroporation of siRNA to Krt19 into Krt18-null TA yielded a strength deficit of 18% after injury compared with controls. Our results suggest that Krt18 plays a complementary role to Krt19 in skeletal muscle in both assembling keratin-based filaments and transducing contractile force. [ABSTRACT FROM AUTHOR]

Mechanical forces regulate muscle development, hypertrophy, and homeostasis. Force-transmitting structures allow mechanotransduction at the sarcolemma, cytoskeleton, and nuclear envelope. There is growing evidence that Yes-associated protein (YAP) serves as a nuclear relay of mechanical signals and can induce a range of downstream signaling cascades. Dystrophin is a sarcolemma-associated protein, and its absence underlies the pathology in Duchenne muscular dystrophy. We tested the hypothesis that the absence of dystrophin in muscle would result in reduced YAP signaling in response to loading. Following in vivo contractile loading in muscles of healthy (wild-type; WT) mice and mice lacking dystrophin (mdx), we performed Western blots of whole and fractionated muscle homogenates to examine the ratio of phospho (cytoplasmic) YAP to total YAP and nuclear YAP, respectively. We show that in vivo contractile loading induced a robust increase in YAP expression and its nuclear localization in WT muscles. Surprisingly, in mdx muscles, active YAP expression was constitutively elevated and unresponsive to load. Results from qRT-PCR analysis support the hyperactivation of YAP in vivo in mdx muscles, as evidenced by increased gene expression of YAP downstream targets. In vitro assays of isolated myofibers plated on substrates with high stiffness showed YAP nuclear labeling for both genotypes, indicating functional YAP signaling in mdx muscles. We conclude that while YAP signaling can occur in the absence of dystrophin, dystrophic muscles have altered mechanotransduction, whereby constitutively active YAP results in a failure to respond to load, which could be attributed to the increased state of "pre-stress" with increased cytoskeletal and extracellular matrix stiffness. [ABSTRACT FROM AUTHOR]

Key points: Breast cancer 1 early onset gene codes for the DNA repair enzyme, breast cancer type 1 susceptibility protein (BRCA1). The gene is prone to mutations that cause a loss of protein function.BRCA1/Brca1 has recently been found to regulate several cellular pathways beyond DNA repair and is expressed in skeletal muscle.Skeletal muscle specific knockout of Brca1 in mice caused a loss of muscle quality, identifiable by reductions in muscle force production and mitochondrial respiratory capacity.Loss of muscle quality was associated with a shift in muscle phenotype and an accumulation of mitochondrial DNA mutations.These results demonstrate that BRCA1 is necessary for skeletal muscle function and that increased mitochondrial DNA mutations may represent a potential underlying mechanism. Recent evidence suggests that the breast cancer 1 early onset gene (BRCA1) influences numerous peripheral tissues, including skeletal muscle. The present study aimed to determine whether induced‐loss of the breast cancer type 1 susceptibility protein (Brca1) alters skeletal muscle function. We induced genetic ablation of exon 11 in the Brca1 gene specifically in the skeletal muscle of adult mice to generate skeletal muscle‐specific Brca1 homozygote knockout (Brca1KOsmi) mice. Brca1KOsmi exhibited kyphosis and decreased maximal isometric force in limb muscles compared to age‐matched wild‐type mice. Brca1KOsmi skeletal muscle shifted toward an oxidative muscle fibre type and, in parallel, increased myofibre size and reduced capillary numbers. Unexpectedly, myofibre bundle mitochondrial respiration was reduced, whereas contraction‐induced lactate production was elevated in Brca1KOsmi muscle. Brca1KOsmi mice accumulated mitochondrial DNA mutations and exhibited an altered mitochondrial morphology characterized by distorted and enlarged mitochondria, and these were more susceptible to swelling. In summary, skeletal muscle‐specific loss of Brca1 leads to a myopathy and mitochondriopathy characterized by reductions in skeletal muscle quality and a consequent kyphosis. Given the substantial impact of BRCA1 mutations on cancer development risk in humans, a parallel loss of BRCA1 function in patient skeletal muscle cells would potentially result in implications for human health. Key points: Breast cancer 1 early onset gene codes for the DNA repair enzyme, breast cancer type 1 susceptibility protein (BRCA1). The gene is prone to mutations that cause a loss of protein function.BRCA1/Brca1 has recently been found to regulate several cellular pathways beyond DNA repair and is expressed in skeletal muscle.Skeletal muscle specific knockout of Brca1 in mice caused a loss of muscle quality, identifiable by reductions in muscle force production and mitochondrial respiratory capacity.Loss of muscle quality was associated with a shift in muscle phenotype and an accumulation of mitochondrial DNA mutations.These results demonstrate that BRCA1 is necessary for skeletal muscle function and that increased mitochondrial DNA mutations may represent a potential underlying mechanism. [ABSTRACT FROM AUTHOR]

Background: Massive rotator cuff tears (RCTs) begin as primary tendon injuries and cause a myriad of changes in the muscle, including atrophy, fatty infiltration (FI), and fibrosis. However, it is unclear which changes are most closely associated with muscle function. Purpose: To determine if FI of the supraspinatus muscle after acute RCT relates to short-term changes in muscle function. Study Design: Controlled laboratory study. Methods: Unilateral RCTs were induced in female rabbits via tenotomy of the supraspinatus and infraspinatus. Maximal isometric force and rate of fatigue were measured in the supraspinatus in vivo at 6 and 12 weeks after tenotomy. Computed tomography scanning was performed, followed by histologic analysis of myofiber size, FI, and fibrosis. Results: Tenotomy resulted in supraspinatus weakness, reduced myofiber size, FI, and fibrosis, but no differences were evident between 6 and 12 weeks after tenotomy except for increased collagen content at 12 weeks. FI was a predictor of supraspinatus weakness and was strongly correlated to force, even after accounting for muscle cross-sectional area. While muscle atrophy accounted for the loss in force in tenotomized muscles with minimal FI, it did not account for the greater loss in force in tenotomized muscles with the most FI. Collagen content was not strongly correlated with maximal isometric force, even when normalized to muscle size. Conclusion: After RCT, muscle atrophy results in the loss of contractile force from the supraspinatus, but exacerbated weakness is observed with increased FI. Therefore, the level of FI can help predict contractile function of torn rotator cuff muscles. Clinical Relevance: Markers to predict contractile function of RCTs will help determine the appropriate treatment to improve functional recovery after RCTs. [ABSTRACT FROM AUTHOR]

The breast cancer type 1 susceptibility protein (Brca1) is a regulator of DNA repair in mammary gland cells; however, recent cell culture evidence suggests that Brca1 influences other processes, including those in nonmammary cells. In this study, we sought to determine whether Brca1 is necessary for metabolic regulation of skeletal muscle using a novel in vivo mouse model. We developed an inducible skeletal muscle-specific Brca1knockout (BRCA1KOsmi) model to test whether Brca1 expression is necessary for maintenance of metabolic function of skeletal muscle when exposed to a high-fat diet (HFD). Our data demonstrated that deletion of Brca1 prevented HFD-induced alterations in glucose and insulin tolerance. Irrespective of diet, BRCA1KOsmi mice exhibited significantly lower ADP-stimulated complex I mitochondrial respiration rates compared to age-matched wild-type (WT) mice. The data show that Brca1 has the ability to localize to the mitochondria in skeletal muscle and that BRCA1KOsmi mice exhibit higher whole-body CO2 production, respiratory exchange ratio, and energy expenditure, compared with the WT mice. Our results demonstrate that loss of Brca1 in skeletal muscle leads to dysregulated metabolic function, characterized by decreased mitochondrial respiration. Thus, any condition that results in loss of Brca1 function could induce metabolic imbalance in skeletal muscle. [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]

Introduction: Dystrophic muscle is particularly susceptible to eccentric contraction-induced injury. We tested the hypothesis that electrical impedance myography (EIM) can detect injury induced by maximal-force lengthening contractions.Methods: We induced injury in the quadriceps of wild-type (WT) and dystrophic (mdx) mice with eccentric contractions using an established model.Results: mdx quadriceps had significantly greater losses in peak twitch and tetany compared with losses in WT quadriceps. Injured muscle showed a significant increase in EIM characteristic frequency in both WT (177 ± 7.7%) and mdx (167 ± 7.8%) quadriceps. EIM also revealed decreased extracellular resistance for both WT and mdx quadriceps after injury.Discussion: Our results show overall agreement between muscle function and EIM measurements of injured muscle, indicating that EIM is a viable tool to assess injury in dystrophic muscle. Muscle Nerve 56: E85-E94, 2017. [ABSTRACT FROM AUTHOR]

Background: Rotator cuff (RTC) tears are a common clinical problem resulting in adverse changes to the muscle, but there is limited information comparing histopathology to contractile function. This study assessed supraspinatus force and susceptibility to injury in the rat model of RTC tear, and compared these functional changes to histopathology of the muscle.Methods: Unilateral RTC tears were induced in male rats via tenotomy of the supraspinatus and infraspinatus. Maximal tetanic force and susceptibility to injury of the supraspinatus muscle were measured in vivo at day 2 and day 15 after tenotomy. Supraspinatus muscles were weighed and harvested for histologic analysis of the neuromuscular junction (NMJ), intramuscular lipid, and collagen.Results: Tenotomy resulted in eventual atrophy and weakness. Despite no loss in muscle mass at day 2 there was a 30% reduction in contractile force, and a decrease in NMJ continuity and size. Reduced force persisted at day 15, a time point when muscle atrophy was evident but NMJ morphology was restored. At day 15, torn muscles had decreased collagen-packing density and were also more susceptible to contraction-induced injury.Conclusion: Muscle size and histopathology are not direct indicators of overall RTC contractile health. Changes in NMJ morphology and collagen organization were associated with changes in contractile function and thus may play a role in response to injury. Although our findings are limited to the acute phase after a RTC tear, the most salient finding is that RTC tenotomy results in increased susceptibility to injury of the supraspinatus. [ABSTRACT FROM AUTHOR]

While excessive tensile strain can be detrimental to nerve function, strain can be a positive regulator of neuronal outgrowth. We used an in vivo rat model of sciatic nerve strain to investigate signaling mechanisms underlying peripheral nerve response to deformation. Nerves were deformed by 11% and did not demonstrate deficits in compound action potential latency or amplitude during or after 6 h of strain. As revealed by Western blotting, application of strain resulted in significant upregulation of mammalian target of rapamycin (mTOR) and S6 signaling in nerves, increased myelin basic protein (MBP) and β-actin levels, and increased phosphorylation of neurofilament subunit H (NF-H) compared with unstrained (sham) contralateral nerves (P β 0.05 for all comparisons, paired two-tailed t-test). Strain did not alter neuron-specific β3-tubulin or overall nerve tubulin levels compared with unstrained controls. Systemic rapamycin treatment, thought to selectively target mTOR complex 1 (mTORC1), suppressed mTOR/S6 signaling, reduced levels of MBP and overall tubulin, and decreased NF-H phosphorylation in nerves strained for 6 h, revealing a role for mTOR in increasing MBP expression and NF-H phosphorylation, and maintaining tubulin levels. Consistent with stretchinduced increases in MBP, immunolabeling revealed increased S6 signaling in Schwann cells of stretched nerves compared with unstretched nerves. In addition, application of strain to cultured adult dorsal root ganglion neurons showed an increase in axonal protein synthesis based on a puromycin incorporation assay, suggesting that neuronal translational pathways also respond to strain. This work has important implications for understanding mechanisms underlying nerve response to strain during development and regeneration. NEW & NOTEWORTHY: Peripheral nerves experience tensile strain (stretch) during development and movement. Excessive strain impairs neuronal function, but moderate strains are accommodated by nerves and can promote neuronal growth; mechanisms underlying these phenomena are not well understood. We demonstrated that levels of several structural proteins increase following physiological levels of nerve strain and that expression of a subset of these proteins is regulated by mTOR. Our work has important implications for understanding nerve development and strain-based regenerative strategies. [ABSTRACT FROM AUTHOR]

Background: Most cultured enzymatically dissociated adult myofibers exhibit spatially uniform (UNI) contractile responses and Ca2+ transients over the entire myofiber in response to electric field stimuli of either polarity applied via bipolar electrodes. However, some myofibers only exhibit contraction and Ca2+ transients at alternating (ALT) ends in response to alternating polarity field stimulation. Here, we present for the first time the methodology for identification of ALT myofibers in primary cultures and isolated muscles, as well as a study of their electrophysiological properties. Results : We used high-speed confocal microscopic Ca2+ imaging, electric field stimulation, microelectrode recordings, immunostaining, and confocal microscopy to characterize the properties of action potential-induced Ca2+ transients, contractility, resting membrane potential, and staining of T-tubule voltage-gated Na+ channel distribution applied to cultured adult myofibers. Here, we show for the first time, with high temporal and spatial resolution, that normal control myofibers with UNI responses can be converted to ALT response myofibers by TTX addition or by removal of Na+ from the bathing medium, with reappearance of the UNI response on return of Na+. Our results suggest disrupted excitability as the cause of ALT behavior and indicate that the ALT response is due to local depolarization-induced Ca2+ release, whereas the UNI response is triggered by action potential propagation over the entire myofiber. Consistent with this interpretation, local depolarizing monopolar stimuli give uniform (propagated) responses in UNI myofibers, but only local responses at the electrode in ALT myofibers. The ALT responses in electrically inexcitable myofibers are consistent with expectations of current spread between bipolar stimulating electrodes, entering (hyperpolarizing) one end of a myofiber and leaving (depolarizing) the other end of the myofiber. ALT responses were also detected in some myofibers within intact isolated whole muscles from wild-type and MDX mice, demonstrating that ALT responses can be present before enzymatic dissociation. Conclusions: We suggest that checking for ALT myofiber responsiveness by looking at the end of a myofiber during alternating polarity stimuli provides a test for compromised excitability of myofibers, and could be used to identify inexcitable, damaged or diseased myofibers by ALT behavior in healthy and diseased muscle. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD), an X-linked disorder caused by the lack of dystrophin, is characterized by the progressive wasting of skeletal muscles. To date, what is known about dystrophin function is derived from studies of dystrophin-deficient animals, with the most common model being the mdx mouse. Most studies on patients with DMD and in mdx mice have focused on skeletal muscle and the development of therapies to reverse, or at least slow, the severe muscle wasting and progressive degeneration. However, dystrophin is also expressed in the CNS. Both mdx mice and patients with DMD can have cognitive and behavioral changes, but studies in the dystrophic brain are limited. We examined the brain structure and metabolites of mature wild type (WT) and mdx mice using magnetic resonance imaging and spectroscopy (MRI/MRS). Both structural and metabolic alterations were observed in the mdx brain. Enlarged lateral ventricles were detected in mdx mice when compared to WT. Diffusion tensor imaging (DTI) revealed elevations in diffusion diffusivities in the prefrontal cortex and a reduction of fractional anisotropy in the hippocampus. Metabolic changes included elevations in phosphocholine and glutathione, and a reduction in γ-aminobutyric acid in the hippocampus. In addition, an elevation in taurine was observed in the prefrontal cortex. Such findings indicate a regional structural change, altered cellular antioxidant defenses, a dysfunction of GABAergic neurotransmission, and a perturbed osmoregulation in the brain lacking dystrophin. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle wasting secondary to repeated muscle damage and inadequate repair. Elevations in intracellular free Ca2+ have been implicated in disease progression, and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1 (SERCA1) overexpression has been shown to ameliorate the dystrophic phenotype in mdx mice. The purpose of this study was to assess the effects of SERCA1 overexpression in the more severe mdx/Utr-/- mouse model of DMD. Mice overexpressing SERCA1 were crossed with mdx/Utr+/- mice to generate mdx/Utr-/-/+SERCA1 mice and compared with wild-type (WT), WT/+SERCA1, mdx/+SERCA1, and genotype controls. Mice were assessed at ~12 wk of age for changes in Ca2+ handling, muscle mass, quadriceps torque, markers of muscle damage, and response to repeated eccentric contractions. SERCA1- overexpressing mice had a two- to threefold increase in maximal sarcoplasmic reticulum Ca2+-ATPase activity compared with WT which was associated with normalization in body mass for both mdx/+SERCA1 and mdx/Utr-/-/+SERCA1. Torque deficit in the quadriceps after eccentric injury was 2.7-fold greater in mdx/Utr-/- vs. WT mice, but only 1.5-fold greater in mdx/Utr-/-/+SERCA1 vs. WT mice, an attenuation of 44%. Markers of muscle damage (% centrally nucleated fibers, necrotic area, and serum creatine kinase levels) were higher in both mdx and mdx/Utr-/- vs. WT, and all were attenuated by overexpression of SERCA1. These data indicate that SERCA1 overexpression ameliorates functional impairments and cellular markers of damage in a more severe mouse model of DMD. These findings support targeting intracellular Ca2+ control as a therapeutic approach for DMD. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy ( DMD), the most common and severe muscular dystrophy, is caused by the absence of dystrophin. Muscle weakness and fragility (i.e., increased susceptibility to damage) are presumably due to structural instability of the myofiber cytoskeleton, but recent studies suggest that the increased presence of malformed/branched myofibers in dystrophic muscle may also play a role. We have previously studied myofiber morphology in healthy wild-type ( WT) and dystrophic ( MDX) skeletal muscle. Here, we examined myofiber excitability using high-speed confocal microscopy and the voltage-sensitive indicator di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8- ANEPPS) to assess the action potential ( AP) properties. We also examined AP-induced Ca2+ transients using high-speed confocal microscopy with rhod-2, and assessed sarcolemma fragility using elastimetry. AP recordings showed an increased width and time to peak in malformed MDX myofibers compared to normal myofibers from both WT and MDX, but no significant change in AP amplitude. Malformed MDX myofibers also exhibited reduced AP-induced Ca2+ transients, with a further Ca2+ transient reduction in the branches of malformed MDX myofibers. Mechanical studies indicated an increased sarcolemma deformability and instability in malformed MDX myofibers. The data suggest that malformed myofibers are functionally different from myofibers with normal morphology. The differences seen in AP properties and Ca2+ signals suggest changes in excitability and remodeling of the global Ca2+ signal, both of which could underlie reported weakness in dystrophic muscle. The biomechanical changes in the sarcolemma support the notion that malformed myofibers are more susceptible to damage. The high prevalence of malformed myofibers in dystrophic muscle may contribute to the progressive strength loss and fragility seen in dystrophic muscles. [ABSTRACT FROM AUTHOR]

Diseases of striated muscle linked to intermediate filament (IF) proteins are associated with defects in the organization of the contractile apparatus and its links to costameres, which connect the sarcomeres to the cell membrane. Here we study the role in skeletal muscle of synemin, a type IV IF protein, by examining mice null for synemin (synm-null). Synm-null mice have a mild skeletal muscle phenotype. Tibialis anterior (TA) muscles show a significant decrease in mean fiber diameter, a decrease in twitch and tetanic force, and an increase in susceptibility to injury caused by lengthening contractions. Organization of proteins associated with the contractile apparatus and costameres is not significantly altered in the synm-null. Elastimetry of the sarcolemma and associated contractile apparatus in extensor digitorum longus myofibers reveals a reduction in tension consistent with an increase in sarcolemmal deformability. Although fatigue after repeated isometric contractions is more marked in TA muscles of synm-null mice, the ability of the mice to run uphill on a treadmill is similar to controls. Our results suggest that synemin contributes to linkage between costameres and the contractile apparatus and that the absence of synemin results in decreased fiber size and increased sarcolemmal deformability and susceptibility to injury. Thus synemin plays a moderate but distinct role in fast twitch skeletal muscle. [ABSTRACT FROM AUTHOR]

R5346 --> 771.8 --> Skeletal muscle experiences mechanical forces that regulate its development, hypertrophy, and homeostasis. YAP (Yes‐associated protein) not only serves as an indicator of nuclear mechanical signals, but can also induce a wide range of downstream signaling cascades. We previously reported increased YAP signaling in healthy (wild type, WT) muscle following isometric contractile loading, and constitutively active YAP signaling in the muscles of mdxmice (dystrophin‐null, murine model of Duchenne muscular dystrophy, or DMD) and aged mice. We tested the hypothesis that increased YAP signaling contributes to the loss of muscle function following eccentric contraction‐induced injury in WT muscle. We also tested the hypothesis that pharmacological inhibition of YAP signaling (verteporfin, 100 mg/kg delivered via intraperitoneal injection every other day immediately following injury for 10 days) improves muscle function following injury. We found increased YAP signaling following eccentric contraction‐induced muscle injury in healthy muscles, indicated by increased gene expression of YAP and its downstream targets. Moreover, pharmacological inhibition of YAP activity resulted in hastened recovery following eccentric contraction injury. Following 50 eccentric contractions to the quadriceps muscles, vehicle (DMSO) treated muscles had a loss of force of 44.8 ± 14.1% and 42.9 ± 17.1% at days 2 and 4 following injury, while verteporfin treated muscles had a loss of force of 13.7 ± 24.7% and 9.05 ± 24.5% at days 2 and 4 following injury. Both groups were fully recovered at 6 days following injury. We conclude that reducing YAP signaling improves muscle function following injury to WT muscle, and could play a significant role in ameliorating muscle pathology in dystrophic and aged muscle. [ABSTRACT FROM AUTHOR]

Background: Duchenne muscular dystrophy (DMD) is characterized clinically by severe, progressive loss of skeletal muscle. The phenotype is much less severe in the mdx mouse model of DMD than that seen in patients with DMD. However, a "critical period" has been described for the mdx mouse, during which there is a peak in muscle weakness and degeneration/regeneration between the 2nd and 5th weeks of life. A number of studies have employed small animal magnetic resonance imaging (MRI) to examine skeletal muscle in various dystrophic models, but such studies represent a snapshot in time rather than a longitudinal view. Results: The in vivo cross-sectional T2-weighted image of the healthy (wild type, WT) muscles is homogeneously dark and this homogeneity does not change with time, as there is no disease. We, and others, have shown marked changes in MRI in dystrophic muscle, with multiple, unevenly distributed focal hyperintensities throughout the bulk of the muscles. Here we monitored an mdx mouse using MRI from 5 to 80 weeks of age. Temporal MRI scans show an increase in heterogeneity shortly after the critical period, at 9 and 13 weeks of age, with a decrease in heterogeneity thereafter. The 4.3-fold increase in percent heterogeneity at week 9 and 13 is consistent with the notion of an early critical period described for mdx mice. Conclusions: Age is a significant variable in quantitative MR studies of the mdx mouse. The mdx mouse is typically studied during the critical period, at a time that most closely mimics the DMD pathology, but the preliminary findings here, albeit based on imaging only one mdx mouse over time, suggest that the changes in MRI can occur shortly after this period, when the muscles are still recovering. [ABSTRACT FROM AUTHOR]

Key points Strength loss induced by lengthening contractions is typically attributed to damaged force-bearing structures within skeletal muscle. Muscle lacking the structural protein dystrophin, as in Duchenne muscular dystrophy, is particularly susceptible to contraction-induced injury., We tested the hypothesis that changes in neuromuscular junctions (NMJs) contribute to strength loss following lengthening contractions in wild-type and in dystrophic skeletal muscle., NMJs in dystrophic ( mdx) mice, the murine model of Duchenne muscular dystrophy, show discontinuous and dispersed motor end-plate morphology. Following lengthening contractions, mdx quadriceps muscles show a greater loss in force, increased neuromuscular transmission failure and decreased electromyographic measures compared to wild-type., Consistent with NMJ disruption as a mechanism contributing to this force loss, only mdx showed increased motor end-plate discontinuity and dispersion of acetylcholine receptor aggregates., Our results indicate that the NMJ in mdx muscle is particularly susceptible to damage, and might play a role in the exacerbated response to injury in dystrophic muscles., Abstract The most common and severe form of muscular dystrophy is Duchenne muscular dystrophy (DMD), a disorder caused by the absence of dystrophin, a structural protein found on the cytoplasmic surface of the sarcolemma of striated muscle fibres. Considerable attention has been dedicated to studying myofibre damage and muscle plasticity, but there is little information to determine if damage from contraction-induced injury occurs at or near the nerve terminal axon. We used α-bungarotoxin to compare neuromuscular junction (NMJ) morphology in healthy (wild-type, WT) and dystrophic ( mdx) mouse quadriceps muscles and evaluated transcript levels of the post-synaptic muscle-specific kinase signalling complex. Our focus was to study changes in NMJs after injury induced with an established in vivo animal injury model. Neuromuscular transmission, electromyography (EMG), and NMJ morphology were assessed 24 h after injury. In non-injured muscle, muscle-specific kinase expression was significantly decreased in mdx compared to WT. Injury resulted in a significant loss of maximal torque in WT (39 ± 6%) and mdx (76 ± 8%) quadriceps, but significant changes in NMJ morphology, neuromuscular transmission and EMG data were found only in mdx following injury. Compared with WT mice, motor end-plates of mdx mice demonstrated less continuous morphology, more disperse acetylcholine receptor aggregates and increased number of individual acetylcholine receptor clusters, an effect that was exacerbated following injury. Neuromuscular transmission failure increased and the EMG measures decreased after injury in mdx mice only. The data show that eccentric contraction-induced injury causes morphological and functional changes to the NMJs in mdx skeletal muscle, which may play a role in excitation-contraction coupling failure and progression of the dystrophic process. [ABSTRACT FROM AUTHOR]

Intermediate filaments (IFs), composed of desmin and keratins, link myofibrils to each other and to the sarcolemma in skeletal muscle. Fast-twitch muscle of mice lacking the IF proteins, desmin and keratin 19 (K19), showed reduced specific force and increased susceptibility to injury in earlier studies. Here we tested the hypothesis that the number of malformed myofibers in mice lacking desmin (Des-/-), keratin 19 (K19-/-), or both IF proteins (double knockout, DKO) is increased and is coincident with altered excitation-contraction (EC) coupling Ca2+ kinetics, as reported for mdx mice. We quantified the number of branched myofibers, characterized their organization with confocal and electron microscopy (EM), and compared the Ca2+ kinetics of EC coupling in flexor digitorum brevis myofibers from adult Des-/-, K19-/-, or DKO mice and compared them to agematched wild type (WT) and mdx myofibers. Consistent with our previous findings, 9.9% of mdx myofibers had visible malformations. Des-/- myofibers had more malformations (4.7%) than K19-/- (0.9%) or DKO (1.3%) myofibers. Confocal and EM imaging revealed no obvious changes in sarcomere misalignment at the branch points, and the neuromuscular junctions in the mutant mice, while more variably located, were limited to one per myofiber. Global, electrically evoked Ca2+ signals showed a decrease in the rate of Ca2+ uptake (decay rate) into the sarcoplasmic reticulum after Ca2+ release, with the most profound effect in branched DKO myofibers (44% increase in uptake relative to WT). Although branched DKO myofibers showed significantly faster rates of Ca2+ clearance, the milder branching phenotype observed in DKO muscle suggests that the absence of K19 corrects the defect created by the absence of desmin alone. Thus, there are complex roles for desmin-based and K19-based IFs in skeletal muscle, with the null and DKO mutations having different effects on Ca2+ reuptake and myofiber branching. [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]

In skeletal muscle fibers, forces must be transmitted between the plasma membrane and the intracellular contractile lattice, and within this lattice between adjacent myofibrils. Based on their prevalence, biomechanical properties and localization, desmin and keratin intermediate filaments (IFs) are likely to participate in structural connectivity and force transmission. We examined the passive load-bearing response of single fibers from the extensor digitorum longus (EDL) muscles of young (3 months) and aged (10months) wild-type, desmin-null, K19-null, and desmin/K19 double-null mice. Though fibers are more compliant in allmutant genotypes compared to wild-type, the structural response of each genotype is distinct, suggesting multiple mechanisms by which desmin and keratin influence the biomechanical properties of myofibers. This work provides additional insight into the influences of IFs on structure-function relationships in skeletal muscle. It may also have implications for understanding the progression of desminopathies and other IF-related myopathies. [ABSTRACT FROM AUTHOR]

In skeletal muscle fibers, forces must be transmitted between the plasma membrane and the intracellular contractile lattice, and within this lattice between adjacent myofibrils. Based on their prevalence, biomechanical properties and localization, desmin and keratin intermediate filaments (IFs) are likely to participate in structural connectivity and force transmission. We examined the passive load-bearing response of single fibers from the extensor digitorum longus (EDL) muscles of young (3 months) and aged (10months) wild-type, desmin-null, K19-null, and desmin/K19 double-null mice. Though fibers are more compliant in allmutant genotypes compared to wild-type, the structural response of each genotype is distinct, suggesting multiple mechanisms by which desmin and keratin influence the biomechanical properties of myofibers. This work provides additional insight into the influences of IFs on structure-function relationships in skeletal muscle. It may also have implications for understanding the progression of desminopathies and other IF-related myopathies. [ABSTRACT FROM AUTHOR]