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Objective A complete and accurate understanding of extraocular muscle function is important to the veterinary care of the avian eye. This is especially true for birds of prey, which rely heavily on vision for survival and yet are prone to ocular injury and disease. To better understand the function of extraocular muscles in birds of prey, we studied extraocular muscle architecture grossly and histologically. Animals studied This sample was composed of two each of the following species: red-tailed hawk (Buteo jamaicensis), Harris's hawk (Parabuteo unicinctus), great horned owl (Bubo virginianus), and barn owl (Tyto alba). Procedures All extraocular muscles were dissected and weighed. To analyze muscle fiber architecture, the superior oblique and quadratus muscles were dissected, weighed, and sectioned at 5 μm thickness in the transverse plane. We calculated the physiologic cross-sectional area and the ratio of muscle mass to predicted effective maximum tetanic tension. Results and conclusions Hawk and owl extraocular muscles exhibit significant physiological differences that play roles in ocular movements and closure of the nictitating membrane. Owls, which do not exhibit extraocular movement, have muscle architecture suited to stabilize the position of a massive, tubular eye that protrudes significantly from the orbit. Hawks, which have a more globose eye that is largely contained within the orbit, do not require as much muscular stability and instead have muscle architecture that facilitates rapid eye movement.

Common additives in plastics such as bisphenol A (BPA) or phthalates like di-(2-ethylhexyl) phthalate (DEHP) are environmental estrogens that have been shown to be endocrine disruptors in some experimental animal models. This project used the C2C12 cell culture model to examine how exposure to BPA or DEHP affects two aspects of skeletal muscle development, the fusion of myoblasts into myotubes and agrin-induced clustering of acetylcholine receptors (AChRs). During myotube formation AChRs cluster spontaneously. Treatment with motor neuron derived agrin increases the frequency of AChR clusters through an agrin signaling pathway that also clusters other postsynaptic components of the neuromuscular synapse. For this project C2C12 cells were exposed to BPA or DEHP while myoblasts fused into myotubes. After exposure to 10 μM BPA or 100 μM DEHP the frequency of agrin-induced AChR clusters decreased. In addition, myotube formation decreased as a higher percentage of nuclei remained in myoblasts. Furthermore, BPA or DEHP reduced the amount of the myogenic regulatory factor myogenin. This suggests that BPA and DEHP decrease AChR clustering by reducing myogenin. Moreover, plastic additives like BPA and DEHP may pose a risk for skeletal muscle development in humans.

Background. Intestinal dysfunction in theob/obmouse model of diabetes mimics that seen clinically.Methods. We determined the effects of a 4-week genistein diet (600 mg genistein/kg food) on intestinal function (contractility, morphology, AChR, and motility) in femaleob/oband lean mice.Results. Contractility of the jejunum in response to incrementally increasing concentrations of KCl was comparable inob/obfemales and lean controls regardless of a genistein-diet. There were no changes in the wall thickness measured. We assessed the number of clusters of AChR in the jejunum wall; AChR were decreased by 48% inob/obmice versus leans, and the genistein diet reversed this. In utilizing a video-imaging system to evaluate gastrointestinal motility, we determined that the distance between consecutive contractile events was significantly increased by 1.87-fold inob/obmice versus leans, and the genistein diet was without effect.Conclusions. These data suggest that slowed intestinal transit in the diabeticob/obmouse may be due in part to decreased AChR and decreased contraction events occurring per unit time. A genistein diet rescues the number of AChR to levels of leans yet did not change the number of contractile events. Feedingob/obmice a genistein-rich diet has potential therapeutic benefits towards improving the debilitating diabetes-related gastrointestinal dysfunction.

Skeletal muscle development is influenced by myogenic regulatory factors, including the expression of MyoD and myogenin. Our objective was to use the C2C12 cell culture model to test the hypothesis that both MyoD and myogenin were required for agrin-induced acetylcholine receptor (AChR) clustering and the fusion of myoblasts into myotubes. We induced fusion of myoblasts into myotubes by switching from growth medium (GM) to differentiation medium (DM). During myotube formation AChRs cluster spontaneously, but treatment with motor neuron derived agrin increases clustering of AChRs and other postsynaptic components of the neuromuscular synapse. We examined the normal expression pattern of MyoD and myogenin in C2C12 cell culture using immunofluorescence. MyoD was highly expressed while myoblasts were in GM, but expression declined within 72 hours after cell cultures were switched to DM. Myogenin expression was low in GM, but increased when cell cultures were switched to DM. Next we used antibodies to decrease MyoD and/or myogenin function. Fluorescence microscopy images were captured and then analyzed to assess agrin-induced AChR clustering with or without antibody treatment. Finally we calculated the proportion of nuclei in myotubes and myoblasts by creating digital overlays of phase contrast and DAPI stained microscopy images. This allowed the comparison of myotube formation with or without antibody treatment. We report that antibody to either MyoD or myogenin decreases the frequency of agrin-induced AChR clustering without affecting myotube formation. We conclude that agrin-induced AChR clustering requires both MyoD and myogenin.

We investigated the effect of ethanol on skeletal muscle development using C2C12 cell culture. The ethanol concentrations of 10 mM, 25 mM, and 100 mM, were tested because plasma samples of alcohol-dependent individuals fall within this range. We assessed two specific events in skeletal muscle development, the fusion of myoblasts to form myotubes and the acetylcholine receptor (AChR) clustering associated with neuromuscular synapse formation. We report that ethanol does not effect myotube formation or the viability of myoblasts or myotubes in C2C12 cell culture. However, ethanol does effect AChR clustering on C2C12 myotubes. As motor neurons approach skeletal muscle during development, agrin is released by motor neurons and induces AChR clustering on muscle fibers. In our experiments, agrin was applied to cell cultures during the period when myoblasts fuse to form myotubes. In cell cultures exposed to ethanol during myotube formation, agrin-induced AChR clustering was decreased compared to untreated cultures. In cell cultures exposed to ethanol during myoblast proliferation, with ethanol removed during myotube formation, agrin-induced AChR clustering was unaffected. We conclude that exposure to a physiologically relevant concentration of ethanol during the specific period of myotube formation decreases agrin-induced AChR clustering.

Background Humans are exposed to nitrate predominantly through diet with peak plasma concentrations within an hour after ingestion, but additional exposure is obtained from the environment, and minimally through de novo synthesis. Higher nitrate consumption has been associated with methemoglobinemia, spontaneous abortions, atherosclerosis, myocardial ischemia, septic and distressed lung, inflammatory bowel disease, amyotrophic lateral sclerosis, and neural tube defects. However, skeletal muscle development has not been examined. Methods C2C12 skeletal muscle cell cultures were maintained, myoblasts were fused into myotubes, and then cultures were exposed to motor neuron derived agrin to enhance acetylcholine receptor (AChR) clustering. Untreated cultures were compared with cultures exposed to sodium nitrate at concentrations ranging from 10 ng/mL–100 μg/mL. Results The results reported here demonstrate that 1 μg/mL sodium nitrate was sufficient to decrease the frequency of agrin-induced AChR clustering without affecting myotube formation. In addition, concentrations of sodium nitrate of 1 μg/mL or 100 μg/mL decreased gene expression of the myogenic transcription factor myogenin and AChR in correlation with the agrin-induced AChR clustering data. Conclusions These results reveal that sodium nitrate decreases the frequency of agrin-induced AChR clustering by a mechanism that includes myogenin and AChR gene expression. As a consequence sodium nitrate may pose a risk for skeletal muscle development and subsequent neuromuscular synapse formation in humans.

Myostatin (growth differentiation factor 8, GDF8) inhibits myogenesis and is a potential therapeutic target for muscular dystrophy and other myopathies. Animals with mutations in myostatin genes ha...

We aimed to characterize jejunal function (contractility, motility and morphology), to better understand the intestinal dysfunction seen in the clinically relevant ob/ob mouse model of diabetes and...

Proteoglycans consist of a protein core complexed to glycosaminoglycan (GAG) side chains, are abundant in skeletal muscle cell membranes and basal lamina, and have important functions in neuromuscular synapse development. Treatment with chlorate results in the undersulfation of heparan sulfate and chondroitin sulfate GAGs in cell culture. In addition, chlorate treatment decreases the frequency of spontaneous acetylcholine receptor (AChR) clustering in skeletal muscle cell culture. AChRs and other molecules cluster to form the postsynaptic component of neuromuscular synapses. Chlorate treatment is shown here to decrease the frequency of agrin-induced AChR clustering and agrin-induced tyrosine phosphorylation of the AChR β-subunit. These data suggest that reduced GAG chain sulfation decreases the frequency of AChR clustering by diminishing the agrin signal transduction pathway.

We studied the effect of the estrogenic pesticide methoxychlor (MXC) on skeletal muscle development using C2C12 muscle cell culture. Various concentrations of MXC or beta-estradiol (E) were added to the culture media. MXC (100 microM) disrupted myoblast fusion into myotubes, but 10 microM MXC or 10 microM E had no effect. Correlated with the diminished size of the myotubes, the clustering of acetylcholine receptors (AChRs) was inhibited by 100 microM MXC, but not by 10 microM MXC or 10 microM E. However, since clusters of AChR receptors did form, the postsynaptic clustering mechanism remained intact. Since E did not disrupt myoblast fusion into myotubes or the clustering of AChRs, we conclude that the abnormality induced by MXC is mediated by a mechanism of action that is independent of E. We believe this to be the first demonstration that MXC induces abnormal effects in the process of muscle development in skeletal muscle cell culture.

To investigate the role of acetylcholine receptors (AChRs) in the aggregation of postsynaptic molecules on muscle cells, we utilized the 1R- genetic variant of C2 muscle cells which has very little expression of AChRs in its cell membrane. On C2 myotubes, AChRs cluster spontaneously, with the frequency of clustering greatly enhanced by motor neuron-derived agrin. Signal transduction events driven by agrin, including the tyrosine phosphorylation of muscle-specific kinase (MuSK) and the AChR beta subunit, have been implicated as requirements of postsynaptic scaffold assembly. We show here that some molecules of the postsynaptic scaffold spontaneously aggregate and colocalize on 1R- myotubes at very low frequency, including an as yet unidentified agrin binding molecule, beta-dystroglycan and MuSK. Agrin is unable to increase the frequency of these aggregations, but does cause tyrosine phosphorylation of MuSK. We conclude that free molecules can associate into aggregates independently of AChRs, but AChRs are required for high-frequency molecular aggregation driven by the agrin signalling pathway. MuSK tyrosine phosphorylation appears to precede a requisite event involving AChRs that aggregates postsynaptic molecules.

The neural factor agrin induces the aggregation of acetylcholine receptors (AChRs) and other synaptic molecules on cultured myotubes. This aggregating activity can be mimicked by experimental manipulations that include treatment with neuraminidase or elevated calcium. We report evidence that neuraminidase and calcium act through the agrin signal transduction pathway. The effects of neuraminidase and calcium on AChR clustering are additive with that of agrin at low concentrations and cosaturating at high concentrations. In addition, like agrin, both neuraminidase and calcium cause rapid tyrosine phosphorylation of the muscle-specific kinase (MuSK) and the AChR-beta subunit. Our results argue that all three agents act directly on components of the same signal transduction complex. We suggest that sialic acids on components of the complex inhibit interactions necessary for signal transduction and that disinhibition can result in activation. In such a model, agrin could activate signal transduction by disinhibition or by circumventing the inhibition.

Acetylcholine receptors (AChRs) and other postsynaptic molecules cluster spontaneously on cultured C2 myotubes. The frequency of clustering is enhanced by neural agrin, neuraminidase, or calcium through a signaling pathway which includes tyrosine phosphorylation of a muscle-specific kinase (MuSK) and the AChR β-subunit. Vicia villosa agglutinin (VVA) lectin, previously shown to potentiate agrin-induced clustering on C2 myotubes, is shown here to also potentiate neuraminidase- and calcium-induced clustering of AChRs, while having no effect on the level of tyrosine phosphorylation of MuSK or the AChR β-subunit. We propose that VVA lectin increases the frequency of AChR clustering through a mechanism that is distinct from agrin signaling, and that may involve α-dystroglycan.

Previous studies have reported that motoneurons from the sixth spinal nerve (C6) innervate the majority of muscle fibers in the rat serratus anterior (SA) muscle. The seventh spinal nerve (C7) innervates a limited number of SA fibers, increasing caudally. This topographic map is partially reestablished following denervation. In the present study, muscle fibers of the SA were stained with monoclonal antibodies for the muscle-specific fast myosin heavy chain (F-MHC) and slow myosin heavy chain (S-MHC) proteins. We found that the majority of fibers in the SA muscle stained for F-MHC antibody, and the percentage of muscle fibers staining for S-MHC antibody increased caudally. When newborn SA muscles were denervated and then reinnervated by the entire long thoracic (LT) nerve or only the C6 branch to the LT nerve, the reinnervated muscle had the normal proportion of muscle fibers expressing S-MHC protein. However, if the LT nerve was crushed and only C7 motoneurons allowed to reinnervate the SA muscle, a greater percentage of muscle fibers stained for S-MHC antibody than normal. We conclude that there is a correlation between muscle fiber type and innervation topography in the SA muscle of the rat.

Previous studies have shown remarkable rostrocaudal selectivity by regenerating motoneurons to the rat serratus anterior (SA) muscle after freezing, crushing, or sectioning the long thoracic (LT) nerve. The LT nerve contains motoneurons from both the sixth and seventh cervical spinal nerves (C6 and C7), with C6 motoneurons as the major source of innervation throughout the muscle, and with C7 motoneurons innervating a larger percentage of muscle fibers caudally than rostrally. To determine if synaptic competition can play a role in neuromuscular topography, both the LT nerve and the branch carrying C6 (rostral) motoneurons to the LT nerve were crushed in newborn rats. This approach provides a temporal advantage to regenerating C7 (caudal) motoneurons. After an initial period during which C7 motoneurons reinnervated a larger proportion of muscle fibers than normal in all SA muscle sectors, C6 motoneurons regained their original proportion of rostral muscle fibers. Caudally, however, C7 motoneurons maintained an expanded territory. With this two-site crush method, the number of C6 motoneurons that reinnervate the SA muscle was significantly decreased from normal, whereas the number of C7 motoneurons remained the same. It is concluded that when C7 motoneurons are given a temporal advantage, synaptic specificity can be altered transiently in rostral muscle sectors and permanently in caudal sectors, and this is correlated with a disproportionate loss of C6 motoneurons. Moreover, this may be an important model for studies of synaptic competition, where terminals destined to be eliminated can be identified beforehand. © 1995 John Wiley & Sons, Inc.

As motor neurons approach skeletal muscle during development, agrin is released and induces acetylcholine receptor (AChR) clustering. Our laboratory investigates the effect of environmental agents on skeletal muscle development by using C2C12 cell culture. For the current project, we investigated both short-term and long-term exposure to caffeine, nicotine, or both, at physiologically relevant concentrations. Short-term exposure was limited to the last 48 h of myotube formation, whereas a long-term exposure of 2 weeks allowed for several generations of myoblast proliferation followed by myotube formation. Both agrin-induced and spontaneous AChR clustering frequencies were assessed. For agrin-induced AChR clustering, agrin was added for the last 16 h of myotube formation. Caffeine, nicotine, or both significantly decreased agrin-induced AChR clustering during short-term and long-term exposure. Furthermore, caffeine, nicotine, or both significantly decreased spontaneous AChR clustering during long-term, but not short-term exposure. Surprisingly, caffeine and nicotine in combination did not decrease AChR clustering beyond the effect of either treatment alone. We conclude that physiologically relevant concentrations of caffeine or nicotine decrease AChR clustering. Moreover, we predict that fetuses exposed to caffeine or nicotine may be less likely to form appropriate neuromuscular synapses.

We studied the effect of the estrogenic pesticide methoxychlor (MXC) on skeletal muscle development using C2C12 cell culture. Myoblast cultures were exposed to various concentrations of MXC at various times during the process of myoblast fusion into myotubes. We observed that MXC exposure decreased myotube formation. In addition, we observed myoblasts with cytoplasmic vacuoles in cultures exposed to MXC. Because cytoplasmic vacuoles can be characteristic of cell death, apoptosis assays and trypan blue exclusion assays were performed. We found no difference in the frequency of apoptosis or in the frequency of cell death for cultures exposed to MXC and untreated cultures. Collectively, these results indicate that MXC exposure decreases myotube formation without causing cell death. In contrast, when cell proliferation was assessed, untreated cultures had a myoblast proliferation rate 50% greater than cultures exposed to MXC. We conclude that MXC decreases myotube formation at least in part by slowing myoblast proliferation. Furthermore, we suggest that direct exposure to MXC could affect skeletal muscle development in animals or humans, in addition to the defects in reproductive development that have previously been reported.

The clustering of acetylcholine receptors (AChRs) in skeletal muscle fibers is a critical event in neuromuscular synaptogenesis. AChRs in concert with other molecules form postsynaptic scaffolds in response to agrin released from motor neurons as motor neurons near skeletal muscle fibers in development. Agrin drives an intracellular signaling pathway that precedes AChR clustering and includes the tyrosine phosphorylation of AChRs. In C2C12 myotube culture, agrin application stimulates the agrin signaling pathway and AChR clustering. Previous studies have determined that the frequency of spontaneous AChR clustering is decreased and AChRs are partially inactivated when bound by the acetylcholine agonist nicotine. We hypothesized that nicotine interferes with AChR clustering and consequent postsynaptic scaffold formation. In the present study, C2C12 myoblasts were cultured with growth medium to stimulate proliferation and then differentiation medium to stimulate fusion into myotubes. They were bathed in a physiologically relevant concentration of nicotine and then subject to agrin treatment after myotube formation. Our results demonstrate that nicotine decreases agrin-induced tyrosine phosphorylation of AChRs and decreases the frequency of spontaneous as well as agrin-induced AChR clustering. We conclude that nicotine interferes with postsynaptic scaffold formation by preventing the tyrosine phosphorylation of AChRs, an agrin signaling event that precedes AChR clustering.

Studies with environmental levels of various metals typically focus on observable neurological symptoms in newborns and adults. Use of the C2C12 skeletal muscle cell line as a developmental model enabled us to test whether environmental insults prevented myotube formation or the assembly of the postsynaptic component of the neuromuscular synapse. Specifically, we asked whether the inorganic metal mercury interfered with the fusion of myoblasts into myotubes, acetylcholine receptor (AChR) clustering, or the agrin signaling events that precede AChR clustering. C2C12 myotubes grown in culture medium containing 10 microM mercuric chloride were morphologically indistinguishable from control myotubes at the light-microscopic level, and myoblasts fused into myotubes normally. However, myotubes pretreated with mercury demonstrated a decreased frequency of AChR clustering induced by agrin and other experimental manipulations. Furthermore, mercury pretreatment decreased the agrin-induced tyrosine phosphorylation of the AChR beta subunit, thus inhibiting the agrin signal transduction pathway. In contrast, mercury failed to decrease the frequency of spontaneous AChR clustering, suggesting that spontaneous AChR clustering differs from agrin-induced AChR clustering in some significant way.