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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