Neuromuscular diseases are often caused by inherited mutations that lead to progressive skeletal muscle weakness and degeneration. In diverse populations of normal healthy mice, we observed correlations between the abundance of mRNA transcripts related to mitochondrial biogenesis, the dystrophin-sarcoglycan complex, and nicotinamide adenine dinucleotide (NAD+) synthesis, consistent with a potential role for the essential cofactor NAD+ in protecting muscle from metabolic and structural degeneration. Furthermore, the skeletal muscle transcriptomes of patients with Duchene’s muscular dystrophy (DMD) and other muscle diseases were enriched for various poly[adenosine 5’-diphosphate (ADP)–ribose] polymerases (PARPs) and for nicotinamide N-methyltransferase (NNMT), enzymes that are major consumers of NAD+ and are involved in pleiotropic events, including inflammation. In the mdx mouse model of DMD, we observed significant reductions in muscle NAD+ levels, concurrent increases in PARP activity, and reduced expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ biosynthesis. Replenishing NAD+ stores with dietary nicotinamide riboside supplementation improved muscle function and heart pathology in mdx and mdx/Utr−/− mice and reversed pathology in Caenorhabditis elegans models of DMD. The effects of NAD+ repletion in mdx mice relied on the improvement in mitochondrial function and structural protein expression (α-dystrobrevin and δ-sarcoglycan) and on the reductions in general poly(ADP)-ribosylation, inflammation, and fibrosis. In combination, these studies suggest that the replenishment of NAD+ may benefit patients with muscular dystrophies or other neuromuscular degenerative conditions characterized by the PARP/NNMT gene expression signatures.

Background Muscle strains are one of the most common injuries treated by physicians. Standard conservative therapy for acute muscle strains usually involves short-term rest, ice, and non-steroidal anti-inflammatory medications, but there is no clear consensus on how to accelerate recovery. Hypothesis Local delivery of platelet-rich plasma (PRP) to injured muscles hastens recovery of function. Study Design Controlled laboratory study. We used an established animal model of injury to test the effects of autologous platelet-rich plasma PRP on recovery of contractile function. Methods In vivo, the tibialis anterior muscles (TA) of anesthetized Sprague-Dawley rats were injured by a single (large strain) lengthening contraction or multiple (small strain) lengthening contractions, both of which result in a significant injury. The TA was injected with either PRP, PPP (platelet-poor plasma, as a sham treatment), or received no treatment. Results Both injury protocols yield a similar loss of force. The PRP only had a beneficial effect at one time point after the single contraction injury protocol. However, PRP had a beneficial effect at several time points after the multiple contraction injury protocol, and resulted in a faster recovery time to full contractile function. The sham injections had no effect compared to no treatment. Conclusion Local delivery of PRP can shorten recovery time after a muscle strain injury. Recovery of muscle from the high repetition protocol has already been shown to require myogenesis, whereas recovery from a single strain does not. This difference in mechanism of recovery may explain why PRP was more effective in the high repetition protocol, as PRP is rich in growth factors that can stimulate myogenesis. Since autologous blood products are safe, PRP may be a useful product to use in clinical treatment of muscle injuries.

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.

Lovering, Richard M., and Patrick G. De Deyne. Contractile function, sarcolemma integrity, and the loss of dystrophin after skeletal muscle eccentric contraction-induced injury. Am J Physiol Cell Physiol 286: C230-C238, 2004. First published October 1, 2003 10.1152/ajpcell.00199.2003.-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 (␣-actinin, desmin, dystrophin, ␤-spectrin), integral membrane (␣-and ␤-dystroglycan, sarcoglycan), and extracellular (laminin, fibronectin) proteins. Immediately after injury, P0 was significantly reduced to 4.23 Ϯ 0.22 N, compared with 8.24 Ϯ 1.34 N in noninjured controls, and EBD was detected intracellularly in 54 Ϯ 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 (␤-spectrin) or not at all (␣-and ␤-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 SKELETAL MUSCLE INJURY is characterized by an immediate loss of the ability to produce force. The cause of this force loss has been attributed to such factors as a defect in excitationcontraction (EC) coupling (55), disruption or loss of forcegenerating structures such as actin and myosin (49), and disruption or loss of force-transmitting structures, such as desmin (6,31). Perhaps the best evidence that disruption of force-bearing structures contributes to strength loss after injury comes from single-fiber studies, where reduction in single-fiber maximal force is observed immediately after eccentric injury (32,33). Although the totality of injury is likely the result of many factors, the purpose of this study was to assess structural defects of the sarcolemma, dystrophin, and dystrophin-associated proteins after a single traumatic muscle injury and to relate these observations to contractile properties.The sarcolemma transmits force and is s...

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 contractioninduced 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. AbstractThe 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. 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.

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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.

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.