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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 < 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 stretch-induced 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.

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease in which weakness, increased susceptibility to muscle injury, and inadequate repair underlie the pathology. While most attention has focused within the muscle fiber, we recently demonstrated significant alterations in the neuromuscular junction (NMJ) morphology and resulting neuromuscular transmission failure (NTF) 24 h after injury in mdx mice (murine model for DMD). Here we determine the contribution of NMJ morphology and NTF to the recovery of muscle contractile function post-injury. NMJ morphology and NTF rates were assessed day 0 (immediately after injury) and days 1, 7, 14 and 21 after quadriceps injury. Eccentric injury of the quadriceps resulted in a significant loss of maximal torque in both WT (39 ± 6 %) and mdx (76 ± 8 %) with a full recovery in WT by day 7 and in mdx by day 21. Post-injury alterations in NMJ morphology and NTF were found only in mdx, were limited to days 0 and 1, and were independent of changes in MuSK or AChR expression. Such early changes at the NMJ after injury are consistent with mechanical disruption rather than newly forming NMJs. Furthermore, we show that the dense microtubule network that underlies the NMJ is significantly reduced and disorganized in mdx compared to WT. These structural changes at the NMJ may play a role in the increased NMJ disruption and the exaggerated loss of nerve-evoked muscle force seen after injury to dystrophic muscles.

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease in which weakness, increased susceptibility to muscle injury, and inadequate repair appear to underlie the pathology. While most attention has focused within the muscle fiber, we recently demonstrated in mdx mice (murine model for DMD) significant morphologic alterations at the motor endplate of the neuromuscular junction (NMJ) and corresponding NMJ transmission failure after injury. Here we extend these initial observations at the motor endplate to gain insight into the pre- vs. postsynaptic morphology, as well as the subsynaptic nuclei in healthy (WT) vs. mdx mice. We quantified the discontinuity and branching of the terminal nerve in adult mice. We report mdx- and age-dependent changes for discontinuity and an increase in branching when compared to WT. To examine mdx- and age-dependent changes in the relative localization of pre- and postsynaptic structures, we calculated NMJ occupancy, defined as the ratio of the footprint occupied by presynaptic vesicles vs. that of the underlying motor endplate. The normally congruent coupling between presynaptic and postsynaptic morphology was altered in mdx mice, independent of age. Finally we found an almost two-fold increase in the number of nuclei and an increase in density (nuclei/area) underlying the NMJ. These outcomes suggest substantial remodeling of the NMJ during dystrophic progression. This remodeling reflects plasticity in both pre- and postsynaptic contributors to NMJ structure, and thus perhaps also NM transmission and muscle function.

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 [Ca.sup.2+] transients, and that S100A1 binds competitively with calmodulin to a canonical S100 binding sequence within the calmodulin-binding domain of the skeletal muscle ryanodine receptor. Using voltage clamped fibers, we found that [Ca.sup.2+] release was suppressed at all test membrane potentials in [S100A1.sup.-/-] fibers. Here we examine the role of S 100A1 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 [Ca.sup.2+] binding and transport processes to calculate sarcoplasmic reticulum [Ca.sup.2+] 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.sup.-/-] 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 [Ca.sup.2+] release flux, resulting in impaired contractile activation and force production in skeletal muscle. S100; excitation-contraction coupling; calcium signaling; muscle 10.1152/ajpcell.00180.2010.

We studied the response of dysferlin-null and control skeletal muscle to large- and 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-kDa 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. inflammation; limb-girdle muscular dystrophy type 2B; Miyoshi myopathy; muscle injury doi: 10.1152/ajpcell.00122.2009

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 ([Ca.sup.2+] transients) and susceptibility to osmotic stress ([Ca.sup.2+] 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 (C57BL10). 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 [Ca.sup.2+] signals (indo-1PE-AM) revealed an EC coupling deficit in myofibers with significant branching. Interestingly, peak amplitude of electrically evoked [Ca.sup.2+] 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 [Ca.sup.2+] concentration (i.e., indo ratio) was seen in malformed vs. normal mdx myofibers. Finally, osmotic stress induced the occurrence of [Ca.sup.2+] 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 [Ca.sup.2+] signaling are evident. calcium; muscular dystrophy; skeletal muscle

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 ([E.sub.2]/T and [E.sub.2]/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 = 0.053), [E.sub.2]/T (-0.71, P < 0.001), [E.sub.2]/FAI (-0.63, P = 0.003), and ACL stiffness near ovulation. With the influences of the other variables controlled, there were significant negative partial rank-order correlations between ACL stiffness and [E.sub.2]/T (-0.72, P < 0.001) and [E.sub.2]/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. androgen receptor; gender differences; immunofluorescence; free androgen index

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[degrees]/s throughout a 90[degrees]-arc of motion. Maximal tetanic tension ([P.sub.o]) 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 ([alpha]-actinin, desmin, dystrophin, [beta]-spectrin), integral membrane ([alpha]- and [beta]-dystroglycan, sarcoglycan), and extracellular (laminin, fibrunectin) proteins. Immediately after injury, Po was significantly reduced to 4.23 [+ or -] 0.22 N, compared with 8.24 [+ or -] 1.34 N in noninjured controls, and EBD was detected intracellularly in 54 [+ or -] 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 ([beta]-spectrin) or not at all ([alpha]- and [beta]-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