Search

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.

Duchenne muscular dystrophy (DMD), caused by the absence of the protein dystrophin, 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 there is little information to determine if damage from contraction-induced injury also occurs at or near the nerve terminal axon. Interestingly, both human patients and the mouse model for DMD (the mdx mouse) present fragmented neuromuscular junction (NMJ) morphology. Studies of mdx mice have revealed presynaptic and postsynaptic abnormalities, nerve terminal discontinuity, as well as increased susceptibility of the NMJ to contraction-induced injury with corresponding functional changes in neuromuscular transmission and nerve-evoked electromyography. Focusing on the NMJ as a contributor to functional deficits in the muscle represents a paradigm shift from the more prevalent myocentric perspectives. Further studies are needed to determine the extent to which the nerve-muscle interaction is disrupted in DMD and the role of the NMJ in the dystrophic progression. This chapter lists the tools needed for nerve terminal and NMJ structural analysis using fluorescence imaging, and provides a step-by-step outline for how to stain, image, and analyze the NMJ in skeletal muscle, with specific attention to mdx muscle.

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.

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

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 Sprague-Dawley rats at 900 degrees /s throughout a 90 degrees -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 (alpha-actinin, desmin, dystrophin, beta-spectrin), integral membrane (alpha- and beta-dystroglycan, sarcoglycan), and extracellular (laminin, fibronectin) proteins. Immediately after injury, P0 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 (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.

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]

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, composed of desmin and of keratins, play important roles in linking contractile elements to each other and to the sarcolemma in striated muscle. We examined the contractile properties and morphology of fast-twitch skeletal muscle from mice lacking keratin 19. Tibialis anterior muscles of keratin-19-null mice showed a small but significant decrease in mean fiber diameter and in the specific force of tetanic contraction, as well as increased plasma creatine kinase levels. Costameres at the sarcolemma of keratin-19-null muscle, visualized with antibodies against spectrin or dystrophin, were disrupted and the sarcolemma was separated from adjacent myofibrils by a large gap in which mitochondria accumulated. The costameric dystrophin-dystroglycan complex, which co-purified with γ-actin, keratin 8 and keratin 19 from striated muscles of wild-type mice, co-purified with γ-actin but not keratin 8 in the mutant. Our results suggest that keratin 19 in fast-twitch skeletal muscle helps organize costameres and links them to the contractile apparatus, and that the absence of keratin 19 disrupts these structures, resulting in loss of contractile force, altered distribution of mitochondria and mild myopathy. This is the first demonstration of a mammalian phenotype associated with a genetic perturbation of keratin 19. [ABSTRACT FROM AUTHOR]

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