Chelliah R. Richmonds

Extraocular muscle is defined by a fundamentally distinct gene expression profile.

Skeletal muscle fibers are defined by patterned covariation of key traits that determine contractile and metabolic characteristics. Although the functional properties of most skeletal muscles result from their proportional content of a few conserved muscle fiber types, some, typically craniofacial, muscles exhibit fiber types that appear to lie outside the common phenotypic range. We analyzed gene expression profiles of three putative muscle classes, limb, masticatory, and extraocular muscle (EOM), in adult mice by high-density oligonucleotide arrays. Pairwise comparisons using conservative acceptance criteria identified expression differences in 287 genes between EOM and limb and/or masticatory muscles. Use of significance analysis of microarrays methodology identified up to 400 genes as having an EOM-specific expression pattern. Genes differentially expressed in EOM reflect key aspects of muscle biology, including transcriptional regulation, sarcomeric organization, excitation-contraction coupling, intermediary metabolism, and immune response. These patterned differences in gene expression define EOM as a distinct muscle class and may explain the unique response of these muscles in neuromuscular diseases.

Markedly enhanced susceptibility to experimental autoimmune myasthenia gravis in the absence of decay-accelerating factor protection

Myasthenia gravis (MG) is an autoimmune neuromuscular transmission disorder characterized by loss of acetylcholine receptors (AChRs) due primarily to the production of anti-AChR autoantibodies. In this study we investigated whether the presence of decay-accelerating factor (DAF or CD55), an intrinsic complement regulator, protects against the development of disease. Experimental autoimmune MG was induced in Daf1(-/-) mice (devoid of neuromuscular DAF protein) and their Daf1(+/+) littermates by injection of rat anti-AChR mAb McAb-3. After twenty-four hours, grip strength assessment revealed that Daf1(-/-) mice exhibited hold times of less than 30 seconds, compared with more than 8 minutes for the Daf1(+/+) controls. The weakness was reversed by edrophonium, consistent with a myasthenic disorder. Immunohistochemistry revealed greatly augmented C3b deposition localized at postsynaptic junctions, and radioimmunoassays showed more profound reductions in AChR levels. Electron microscopy demonstrated markedly greater junctional damage in the Daf1(-/-) mice compared with the Daf1(+/+) littermates. Control studies showed equivalent levels of other cell surface regulators, i.e., Crry and CD59. The results demonstrate that mice that lack DAF are markedly more susceptible to anti-AChR-induced MG, which simulates the primary mechanism in the human disease, and strongly suggest that in disease flares complement inhibitors might have therapeutic value.

Constitutive properties, not molecular adaptations, mediate extraocular muscle sparing in dystrophic mdx mice

Extraocular muscle (EOM) is spared in Duchenne muscular dystrophy. Here, we tested putative EOM sparing mechanisms predicted from existing dystrophinopathy models. Data show that mdx mouse EOM contains dystrophin‐glycoprotein complex (DGC)‐competent and DGC‐deficient myofibers distributed in a fiber type‐specific pattern. Up‐regulation of a dystrophin homologue, utrophin, mediates selective DGC retention. Counter to the DGC mechanical hypothesis, an intact DGC is not a precondition for EOM sarcolemmal integrity, and active adaptation at the level of calcium homeostasis is not mechanistic in protection. A partial, fiber type‐specific retention of antiischemic nitric oxide to vascular smooth muscle signaling is not a factor in EOM sparing, because mice deficient in dystrophin and α‐syntrophin, which localizes neuronal nitric oxide synthase to the sarcolemma, have normal EOMs. Moreover, an alternative transmembrane protein, α7β1 integrin, does not appear to substitute for the DGC in EOM. Finally, genomewide expression profiling showed that EOM does not actively adapt to dystrophinopathy but identified candidate genes for the constitutive protection of mdx EOM. Taken together, data emphasize the conditional nature of dystrophinopathy and the potential importance of nonmechanical DGC roles and support the hypothesis that broad, constitutive structural cell signaling, and/or biochemical differences between EOM and other skeletal muscles are determinants of differential disease responsiveness.

Novel complement inhibitor limits severity of experimentally myasthenia gravis

Complement mediated injury of the neuromuscular junction is considered a primary disease mechanism in human myasthenia gravis and animal models of experimentally acquired myasthenia gravis (EAMG). We utilized active and passive models of EAMG to investigate the efficacy of a novel C5 complement inhibitor rEV576, recombinantly produced protein derived from tick saliva, in moderating disease severity.

Hypoglossal and phrenic motoneuron responses to serotonergic active agents in rats

5-Hydroxytryptamine (serotonin, 5-HT) affects upper airway and chest wall inspiratory muscle control. The purpose of this study was to investigate the relative interaction of serotonergic agents on these two muscle groups. We measured the responses of the hypoglossal and phrenic nerves to the systemic administration of serotonergic-active agents and determined the receptor types through which these agents act in anesthetized, vagotomized, paralyzed and artificially ventilated rats. The serotonin precursor, L-5-hydroxytryptophan (L-5-HTP) produced equivalent stimulation of phasic inspiratory activity of the hypoglossal and phrenic nerves. General serotonin antagonists produced significant and equivalent diminution of both motoneuron pools. Specific 5-HT1A stimulation and 5-HT1C/2 antagonism enhanced ventilatory activity. We conclude: (1) a baseline level of serotonergic input to hypoglossal and phrenic motoneuron pools was present, (2) different 5-HT receptors had different effects on ventilatory neural activity, and (3) hypoglossal and phrenic motoneuron pools responded similarly to the serotonergic agents given.

Deficiency of decay accelerating factor and CD59 leads to crisis in experimental myasthenia.

In myasthenia gravis (MG), neuromuscular transmission is disrupted due to the production of autoantibodies against acetylcholine receptors (AChR). In previous work, we showed that decay accelerating factor (DAF or CD55), an intrinsic cell surface complement regulator that disables C3/C5 amplification convertases, protects against receptor loss and muscle weakness. In this study, we examined whether, and if so, to what extent CD59, a downstream intrinsic cell surface regulator that prevents assembly of membrane attack complexes (MACs), contributes to this protection. Twenty-four hours after anti-AChR injection, we found that CD59a-/- mice did not significantly differ from WTs, all Daf1-/- CD59a-/- mice either died or required euthanasia. At 48h, Daf1-/- were significantly weaker than CD59a-/- and WT mice, and for these mice immunohistochemistry revealed marked C9 deposition at postsynaptic junctions, radioimmunoassays showed reductions in AChR levels, and electron microscopy demonstrated massive junctional damage. These data indicate that DAF serves as the initial shield that protects the neuromuscular junction whereas CD59 is a further barrier. They argue that complement inhibitor, particularly if targeted to the receptor, could then have therapeutic value in human MG.

Nitric oxide synthase in aging rat skeletal muscle.

The neuronal isoform of nitric oxide synthase (NOS) is expressed at high concentrations in skeletal muscle, and NO influences muscle contractility, glucose utilization, and free radical damage or protection. NOS activity and expression was evaluated in extensor digitorum longus (EDL), soleus, and diaphragm of 8 and 24 month old Fisher 344 rats. In 8-month-old animals, NOS activity was highest in EDL, which contained the highest percentage of NOS containing fibers, and was lowest in soleus. NOS activity and percentage of NOS containing fibers was significantly reduced in all muscle groups with age. To determine if NOS reduction correlated with free radical injury the level of lipid peroxidation, as measured by malonaldehyde equivalents, was determined. With age lipid peroxidation increased in EDL, was reduced in diaphragm, and showed a non-significant change in soleus. Therefore, a straightforward reduction of NOS activity does not correlate with lipid peroxidation. The reduction of NOS with age in skeletal muscle may be most significant for muscle metabolism and force production and be of limited significance for free radical metabolism.

Nitric oxide synthase expression and effects of nitric oxide modulation on contractility of rat extraocular muscle

Extraocular muscles (EOMs) are specialized skeletal muscles that are constantly active, generate low levels of force for cross sectional area, have rapid contractile speeds, and are highly fatigue resistant. The neuronal isoform of nitric oxide synthase (nNOS) is concentrated at the sarcolemma of fast‐twitch muscles fibers, and nitric oxide (NO) modulates contractility. This study evaluated nNOS expression in EOM and the effect of NO modulation on lateral rectus muscles contractility. nNOS activity was highest in EOM compared with diaphragm, extensor digitorum longus, and soleus. Neuronal NOS was concentrated to the sarcolemma of orbital and global singly innervated fibers, but not evident in the multi‐innervated fibers. The NG‐nitro‐L‐arginine methyl ester (L‐NAME, a NOS inhibitor), increased submaximal tetanic and peak twitch forces. The NO donors S‐nitroso‐N‐acetylcys‐teine (SNAC) and spermineNONOate reduced submaximal tetanic and peak twitch forces. The effect of NO on the contractile force of lateral rectus muscle is greater than previously observed on other skeletal muscle. NO appears more important in modulating contraction of EOM compared with other skeletal muscles, which could be important for the EOMs specialized role in generation of eye movements.—Richmonds, C. R., Kaminski, H. J. Nitric oxide synthase expression and effects of nitric oxide modulation on contractility of rat extraocular muscle. FASEB J. 15, 1764–1770 (2001)

Extraocular Muscle Fatigue

Extraocular muscles (EOM) are particularly resistant to fatigue as determined by functional measures in humans1 and studies of isolated muscle preparations.2 Fatigue resistance in vivo likely stems from EOM’s high vascularity and mitochondrial content. Gene expression profiling also suggests that EOM rely on glucose uptake from blood rather than glycogen breakdown, as occurs in other skeletal muscle.3 The high level of nitric oxide synthase and nitric oxide’s effects on muscle contractility and glucose uptake may further contribute to EOM’s fatigue resistance.4 To further evaluate fatigue properties of EOM, we compared the effects of fatigue protocols and temperature on in vitro tetanic contractions of lateral rectus (LR) and extensor digitorum longus (EDL) of mouse. Both these muscles share properties of rapid contractile speeds. At the end of a 5-minute fatigue regime, LR retained 58.5% of the original force (from 2.323 to 1.359 Newtons/cm2), whereas EDL had only 16.93% (3.775 to 0.639 Newtons/cm2). A significant decrease in force was already observed after 2 minutes. In temperature studies, LR showed a significant increase in force at 27°C compared to 37°C. In contrast, force generation of EDL was decreased at 27°C. Both the muscles exhibited a decrease in force at 20°C compared to higher temperatures. In contrast to EDL, LR maintains and, in fact, increases tetanic force levels after cooling. The studies confirm work in other animals that show EOM to be particularly fatigue resistant.2 The maintenance of tetanic force with cooling of LR suggests more efficient excitation-contraction coupling compared to EDL. The main effect of temperature on muscle function concerns the process of Ca2+ release and uptake. The response of the LR may be due to the small myofibrils, unique troponin isoform composition, abundant mitochondria and sarcoplasmic reticulum, high SR Ca2+ and Ca2+-ATPase found in EOM. EOM may modulate intracellular free Ca2+ levels by both passive and active mechanisms since most EOM fibers are high in Ca2+ binding proteins and mainly express the fast isoform of Ca2+-ATPase.5 The differences in calcium characteristics may be particularly important in understanding the resistance of EOM to degeneration in muscular dystrophy, which may be in part mediated by calcium overload.6

Nitric oxide myotoxicity is age related.

Nitric oxide (NO) is generated under normal conditions in skeletal muscle and acts as a messenger that influences contractility, blood flow, and glucose metabolism. Excess NO generation may occur in pathological states, in particular inflammatory conditions. We demonstrate that incubation of rat extensor digitorum longus muscle with the NO donor, S-nitrosocysteine, leads to release of creatine kinase, a marker of muscle injury after a delay of 90 min. Muscle of old animals was more sensitive to the NO donor. Light microscopic analysis does not show abnormalities, with the exception of an increase in interfiber distance. Histological staining identified no pathological elevations of calcium. The study demonstrates the direct toxicity of NO to skeletal muscle, and that muscle of older animals is differentially susceptible to NO toxicity.