Complex Correlations Between Desmin Content, Myofiber Types, and Innervation Patterns in the Human Extraocular Muscles.

Purpose: To investigate whether the distribution of intermediate filament protein desmin is related to the different patterns of innervation in the human extraocular muscles (EOMs). Methods: EOM samples were analyzed with immunohistochemistry using antibodies against desmin, vimentin, different myosin heavy chain (MyHC) isoforms, and fetal and adult acetylcholine receptor (AChR) subunits. Neuromuscular junctions (NMJs) were identified with α-bungarotoxin or with antibodies against neurofilament and synaptophysin. Results: Desmin was present in the vast majority of myofibers, but it was weakly present or absent in a limited area in the close vicinity of the single en plaque NMJs in less than half of these myofibers. Desmin was either present or lacking in MyHCsto/I myofibers displaying multiple en grappe endings but present in MyHCsto/I myofibers receiving spiral nerve endings. In MyHCeom myofibers displaying multiterminal en plaque endings, desmin was either present or absent irrespective of AChR subunits or EOM layer. Vimentin did not substitute for the lack of desmin. Conclusions: The results indicate that the human EOMs have a more complex cytoskeletal organization than other muscles and suggest additional signalling mechanisms from the NMJs to the myofibers.

The superfast human extraocular myosin is kinetically distinct from the fast skeletal IIa, IIb, and IId isoforms.

Humans express five distinct myosin isoforms in the sarcomeres of adult striated muscle (fast IIa, IId, the slow/cardiac isoform I/ß, the cardiac specific isoform α, and the specialized extraocular muscle isoform). An additional isoform, IIb, is present in the genome but is not normally expressed in healthy human muscles. Muscle fibers expressing each isoform have distinct characteristics including shortening velocity. Defining the properties of the isoforms in detail has been limited by the availability of pure samples of the individual proteins. Here we study purified recombinant human myosin motor domains expressed in mouse C2C12 muscle cells. The results of kinetic analysis show that among the closely related adult skeletal isoforms, the affinity of ADP for actin·myosin (K(AD)) is the characteristic that most readily distinguishes the isoforms. The three fast muscle myosins have K(AD) values of 118, 80, and 55 µM for IId, IIa, and IIb, respectively, which follows the speed in motility assays from fastest to slowest. Extraocular muscle is unusually fast with a far weaker K(AD) = 352 µM. Sequence comparisons and homology modeling of the structures identify a few key areas of sequence that may define the differences between the isoforms, including a region of the upper 50-kDa domain important in signaling between the nucleotide pocket and the actin-binding site.

Anatomical characterization of a rabbit cerebellar eyeblink premotor pathway using pseudorabies and identification of a local modulatory network in anterior interpositus.

Rabbit eyeblink conditioning is a well characterized model of associative learning. To identify specific neurons that are part of the eyeblink premotor pathway, a retrograde transsynaptic tracer (pseudorabies virus) was injected into the orbicularis oculi muscle. Four time points (3, 4, 4.5, and 5 d) were selected to identify sequential segments of the pathway and a map of labeled structures was generated. At 3 d, labeled first-order motor neurons were found in dorsolateral facial nucleus ipsilaterally. At 4 d, second-order premotor neurons were found in reticular nuclei, and sensory trigeminal, auditory, vestibular, and motor structures, including contralateral red nucleus. At 4.5 d, labeled third-order premotor neurons were found in the pons, midbrain, and cerebellum, including dorsolateral anterior interpositus nucleus and rostral fastigial nucleus. At 5 d, labeling revealed higher-order premotor structures. Labeled fourth-order Purkinje cells were found in ipsilateral cerebellar cortex in cerebellar lobule HVI and in lobule I. The former has been implicated in eyeblink conditioning and the latter in vestibular control. Labeled neurons in anterior interpositus were studied, using neurotransmitter immunoreactivity to classify individual cell types and delineate their interconnectivity. Labeled third-order premotor neurons were immunoreactive for glutamate and corresponded to large excitatory projection neurons. Labeled fourth-order premotor interneurons were immunoreactive for GABA (30%), glycine (18%), or both GABA and glycine (52%) and form a functional network within anterior interpositus involved in modulation of motor commands. These results identify a complete eyeblink premotor pathway, deep cerebellar interconnectivity, and specific neurons responsible for the generation of eyeblink responses.

Rhabdomyosarcoma metastases to all extraocular muscles.

A 24-year-old woman developed acute bilateral proptosis. She had a history of rhabdomyosarcoma of the left orbit treated 2 years previously with chemotherapy and radiation. Computed tomography demonstrated enlargement of each of the extraocular muscles in both orbits. Extraocular muscle biopsy confirmed rhabdomyosarcoma. She was treated with radiation but died 2 months after presentation.

Atypical MRI features of intraorbital metastatic melanoma.

A 78-year-old man with a history of melanoma presented with a 2-week history of diplopia, pain, and intermittent blurriness in his right eye. Imaging showed a multicystic mass within the right lateral rectus muscle that was biopsy-proven metastatic melanoma. To our knowledge, this is the first case report of orbital metastasis from melanoma presenting as a multicystic mass intrinsic to the extraocular muscle with layering fluid-fluid levels.

Nebulin isoforms of extraocular muscle.

The extraocular muscles (EOMs), which are responsible for reflexive and voluntary eye movements, have many unique biochemical, physiological, and ultrastructural features that set them apart from other skeletal muscles. For example, rodent EOMs lack M-lines and express EOM-specific myosin heavy chain (MYH13) and alpha-cardiac myosin heavy chain. Recent gene-expression profiling studies indicate the presence of other cardiac-specific proteins in adult EOMs. This interesting mixture of myofibrillar and cytoskeletal proteins poses the questions as to whether nebulette, as opposed to nebulin, might be expressed in EOM, and what isoforms of titin are expressed in the EOM. We have performed gel electrophoresis and immunological analyses to determine the titin and nebulin isoforms expressed in the EOM. We have found that the mass of the titin isoforms expressed in the EOM most closely resemble those found in the skeletal muscles tested, viz., the soleus and extensor digitorum longus (EDL). We also demonstrate that, although the EOM expresses cardiac isoforms of myosin, it does not express nebulette and contains a nebulin isoform with a mass consistent with that found in the prototypical fast hindlimb muscle EDL.

Orbital-conjunctival glomangiomas involving two ocular rectus muscles.

PURPOSE: To report two glomangiomas in one orbit, each isolated to a rectus muscle. DESIGN: Clinicopathologic correlation. METHODS: A 12-year-old boy developed two separate vascular tumors, near the insertions of the medial rectus and superior rectus muscles, respectively. A biopsy of one tumor was studied by light microscopy and immunohistochemistry. RESULTS: Histopathology revealed blood vessels surrounded by cuboidal cells characteristic of glomangioma. The cells showed immunoreactivity for smooth muscle actin and vimentin, supporting the diagnosis. CONCLUSIONS: Glomangioma can involve the rectus muscles in the conjunctiva and orbit, and should be considered in differential diagnosis of vascular tumors in the ocular region.

Utrophin is lacking at the neuromuscular junctions in the extraocular muscles of normal cat: artefact or true?

Extraocular muscles (EOM) are typically spared in Duchenne muscular dystrophy. We hypothesized that this might be due to different patterns of utrophin expression. The expression of utrophin was examined in EOM of normal cats using immunohistochemical methods and Western blot. For detecting acetylcholine receptors (AChR), we used alpha-bungarotoxin. Surprisingly, alpha-bungarotoxin failed to stain the AChR and no expression of utrophin could be detected at the neuromuscular junctions. Our study could indicate that the expression of utrophin is dependent on the structure of the AChR.

Conserved and muscle-group-specific gene expression patterns shape postnatal development of the novel extraocular muscle phenotype.

Current models in skeletal muscle biology do not fully account for the breadth, causes, and consequences of phenotypic variation among skeletal muscle groups. The muscle allotype concept arose to explain frank differences between limb, masticatory, and extraocular (EOM) muscles, but there is little understanding of the developmental regulation of the skeletal muscle phenotypic range. Here, we used morphological and DNA microarray analyses to generate a comprehensive temporal profile for rat EOM development. Based upon coordinate regulation of morphologic/gene expression traits with key events in visual, vestibular, and oculomotor system development, we propose a model that the EOM phenotype is a consequence of extrinsic factors that are unique to its local environment and sensory-motor control system, acting upon a novel myoblast lineage. We identified a broad spectrum of differences between the postnatal transcriptional patterns of EOM and limb muscle allotypes, including numerous transcripts not traditionally associated with muscle fiber/group differences. Several transcription factors were differentially regulated and may be responsible for signaling muscle allotype specificity. Significant differences in cellular energetic mechanisms defined the EOM and limb allotypes. The allotypes were divergent in many other functional transcript classes that remain to be further explored. Taken together, we suggest that the EOM allotype is the consequence of tissue-specific mechanisms that direct expression of a limited number of EOM-specific transcripts and broader, incremental differences in transcripts that are conserved by the two allotypes. This represents an important first step in dissecting allotype-specific regulatory mechanisms that may, in turn, explain differential muscle group sensitivity to a variety of metabolic and neuromuscular diseases.

Expression of Trk receptors in the oculomotor system of the adult cat.

We examined the expression of the three Trk receptors for neurotrophins (TrkA, TrkB, and TrkC) in the extraocular motor nuclei of the adult cat by using antibodies directed against the full-Trk proteins in combination with horseradish peroxidase retrograde tracing. The three receptors were present in all neuronal populations investigated, including abducens motoneurons and internuclear neurons, medial rectus motoneurons of the oculomotor nucleus, and trochlear motoneurons. They were also present in the vestibular and prepositus hypoglossi nuclei. TrkA, TrkB, and TrkC immunopositive cells were found in similar percentages in the oculomotor and in the trochlear nuclei. In the abducens nucleus, however, a significantly higher percentage of cells expressed TrkB than the other two receptors, among both motoneurons (81.8%) and internuclear neurons (88.4%). The percentages obtained for the three Trk receptors in identified neuronal populations pointed to the colocalization of two or three receptors in a large number of cells. We used confocal microscopy to elucidate the subcellular location of Trk receptors. In this case, abducens motoneurons and internuclear neurons were identified with antibodies against choline acetyltransferase and calretinin, respectively. We found a different pattern of staining for each neurotrophin receptor, suggesting the possibility that each receptor and its cognate ligand may use a different route for cellular signaling. Therefore, the expression of Trk receptors in oculomotor, trochlear, and abducens motoneurons, as well as abducens internuclear neurons, suggests that their associated neurotrophins may exert an influence on the normal operation of the oculomotor circuitry. The presence of multiple Trk receptors on individual cells indicates that they likely act in concert with each other to regulate distinct functions.

In situ expression of thyrotropin receptor in the extraocular muscles of patients with thyroid-associated ophthalmopathy.

OBJECTIVE: To investigate the expression of thyrotropin receptor (TSHR) in the extraocular muscles (EOM) of the eyes of patients with thyroid-associated ophthalmopathy (TAO). METHODS: EOM specimens were obtained from 12 patients with severe TAO and 10 healthy subjects within 1 hour after accidental death and the paraffin-embedded sections were observed by streptavidin-peroxidase (SP) immunohistochemical methods. RESULT: The expression of TSHR was observed in the perimysium, cytomembrane and cytoplasm in 10 of the 12 TAO specimens, accounting for a TSHR expression rate of 83.3% in the EOM of TAO patients. No TSHR protein was found in the healthy subjects. CONCLUSION: High TSHR expression in the EOM of TAO patients indicates that as a common antigen between the orbit and thyroid, TSHR play a key role in the pathogenesis of TAO.

Myosin light chain 1 isoforms in slow fibers from global and orbital layers of canine rectus muscles.

PURPOSE: Initial results of an examination of the low molecular mass (< or =45 kDa) protein composition of canine rectus muscle homogenates, based on gel electrophoresis, revealed a distinct difference between the global and orbital layers in the myosin light chain (MLC)-1 region. The objectives of the present study were, therefore, to identify isoforms of MLC1 in homogenates of the global and orbital layers of adult canine rectus muscles and to determine the MLC1 isoform expression pattern among single muscle fibers isolated from both layers. METHODS: Muscle homogenates and single fibers from the global and orbital layers of canine rectus muscles were analyzed, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) was used to identify a protein band in the orbital layer that comigrated with MLC1 in the adult canine atrium. RESULTS: Adult canine extraocular rectus muscles expressed embryonic skeletal/atrial MLC1 (MLC1(E/A)), in addition to the fast-type MLC1 (MLC1F) and slow-type MLC1 (MLC1S) isoforms expressed in limb skeletal muscles. MLC1(E/A) was detected in slow fibers of the orbital but not the global layer, and MLC1S was detected in slow fibers in only the global but not the orbital layer. Densitometric analysis of gel bands from homogenates supported these results, with significantly greater amounts of MLC1S in the global layer and of MLC1(E/A) in the orbital layer. CONCLUSIONS: MLC1(E/A) is expressed in rectus muscles of adult dogs. Furthermore, two types of slow fibers, distinguished on the basis of MLC1 isoform expression, exist in separate layers of canine rectus muscles.

Intercellular adhesion molecule 1 gene polymorphisms in Graves' disease.

It was recently suggested that genetic factors could play a major role in the development of Graves' disease (GD). The aim of the present study was to evaluate the frequency of the c.721G-->A polymorphism and the c.1405A-->G polymorphism of the intercellular adhesion molecule 1 (ICAM-1) gene in subjects with GD compared with that in healthy controls, because ICAM-1 was found to play a key role in lymphocyte infiltration into the thyroid gland and the concentration of the soluble form of ICAM-1 correlates significantly with the clinical activity and treatment status in GD. We have analyzed the association of ICAM-1 polymorphisms with the age at onset of GD and the presence of ophthalmopathy. In a group of 235 patients with GD and 211 healthy controls we have shown that polymorphism at position c.721G-->A is associated with an earlier age of GD onset and that the c.1405A-->G polymorphism of the ICAM-1 gene could predispose to Graves' ophthalmopathy. This suggests that G241R and K469E amino acid substitutions in the ICAM-1 molecule could influence the intensity/duration of the autoimmunity process and the infiltration of orbital tissues. It could be speculated that therapy that modulates ICAM-1 function may delay the onset and/or prolong the remission and/or have an influence on clinical manifestations of GD.

The superfast extraocular myosin (MYH13) is localized to the innervation zone in both the global and orbital layers of rabbit extraocular muscle.

Extraocular muscles (EOMs) are the most molecularly heterogeneous and physiologically diverse mammalian striated muscles. They express the entire array of striated muscle myosins, including a specialized myosin heavy chain MYH13, which is restricted to extraocular and laryngeal muscles. EOMs also exhibit a breadth of contractile activity, from superfast saccades to slow tracking and convergence movements. These movements are accomplished by the action of six ultrastructurally defined fiber types that differ from the type IIa, IIb, IIx and I fibers found in other skeletal muscles. Attempts to associate different eye movements with either the expression of different myosins or the activity of particular EOM fiber types are complicated by the molecular heterogeneity of several of the fiber types, and by electromyography studies showing that the majority of extraocular motor units participate in both fast and slow eye movements. To better understand the role of MYH13 in ocular motility, we generated MYH13-sequence-specific antibodies and used SDS-PAGE to quantify the regional distribution of myosin in EOM and to characterize its heterogeneity in single fibers. These studies demonstrate that MYH13 is preferentially expressed in the majority of orbital and global fibers in the central innervation zone of rabbit EOM. Many individual fibers express MYH13 with the fast IIb myosin and varying amounts of IIx myosin. The differential localization of MYH13, coupled with specialization of the sarcoplasmic reticulum and thin filament systems, probably explains how activation of the endplate band region enables the majority of EOM fibers to contribute to superfast contractions.

Oligomerization of polyalanine expanded PABPN1 facilitates nuclear protein aggregation that is associated with cell death.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. The autosomal dominant form of this disease is caused by short expansions of a (GCG)(6) repeat to (GCG)(8-13) in the PABPN1 gene, which results in the expansion of a polyalanine stretch from 10 to 12-17 alanines in the N-terminus of the protein. Mutated PABPN1 (mPABPN1) is able to induce nuclear protein aggregation and form filamentous nuclear inclusions, which are the pathological hallmarks of OPMD. PABPN1, when bound to poly(A) RNA, forms both linear filaments and discrete-sized, compact oligomeric particles in vitro. In the absence of poly(A) RNA, PABPN1 can form oligomers. Here we report that: (i) oligomerization of PABPN1 is mediated by two potential oligomerization domains (ODs); (ii) inactivating oligomerization of mPABPN1 by deletions of 6-8 amino acids in either of the ODs prevents nuclear protein aggregation; (iii) expression of mPABPN1 in COS-7 cells is associated with cell death; and (iv) preventing nuclear protein aggregation by inactivating oligomerization of mPABPN1 significantly reduces cell death. These findings suggest that oligomerization of PABPN1 plays a crucial role in the formation of OPMD nuclear protein aggregation, while the expanded polyalanine stretch is necessary but not sufficient to induce OPMD protein aggregation, and that the nuclear protein aggregation might be toxic and cause cell death. These observations also imply that inactivation of oligomerization of mPABPN1 might be a useful therapeutic strategy for OPMD.

Analysis of human sarcospan as a candidate gene for CFEOM1.

BACKGROUND: Congenital fibrosis of the extraocular muscles type 1 (CFEOM1) is an autosomal dominant eye movement disorder linked to the pericentromere of chromosome 12 (12p11.2 - q12). Sarcospan is a member of the dystrophin associated protein complex in skeletal and extraocular muscle and maps to human chromosome 12p11.2. Mutations in the genes encoding each of the other components of the skeletal muscle sarcospan-sarcoglycan complex (alpha - delta sarcoglycan) have been shown to cause limb girdle muscular dystrophy (LGMD2C-F). To determine whether mutations in the sarcospan gene are responsible for CFEOM1 we: (1) attempted to map sarcospan to the CFEOM1 critical region; (2) developed a genomic primer set to directly sequence the sarcospan gene in CFEOM1 patients; and (3) generated an anti-sarcospan antibody to examine extraocular muscle biopsies from CFEOM1 patients. RESULTS: When tested by polymerase chain reaction, sarcospan sequence was not detected on yeast or bacterial artificial chromosomes from the CFEOM1 critical region. Sequencing of the sarcospan gene in CFEOM1 patients from 6 families revealed no mutations. Immunohistochemical studies of CFEOM1 extraocular muscles showed normal levels of sarcospan at the membrane. Finally, sarcospan was electronically mapped to bacterial artificial chromosomes that are considered to be outside of the CFEOM1 critical region. CONCLUSIONS: In this report we evaluate sarcospan as a candidate gene for CFEOM1. We have found that it is highly unlikely that sarcospan is involved in the pathogenesis of this disease. As of yet no sarcospan gene mutations have been found to cause muscular abnormalities.

Early specialization of the superfast myosin in extraocular and laryngeal muscles.

Extraocular muscle (EOM) exhibits high-velocity, low-tension contractions compared with other vertebrate striated muscles. These distinctive properties have been associated with a novel myosin heavy chain (MyHC) isoform, MyHC-EO. An atypical MyHC, MyHC IIL, has also been identified in laryngeal muscles that have similarly fast contractile properties. It co-migrates with MyHC-EO on high-resolution SDS gels, but appeared to be encoded by a different mRNA. We combined CNBr peptide maps and full-length cDNA sequences to show that rabbit muscle EO and IIL MyHCs are identical. Analysis of the 5; untranslated region (5;UTR) of the mRNAs identified three variants that result from a combination of alternative splicing and multiple transcription initiation sites. This complex pattern of 5;UTRs has not been reported previously for MyHC genes. We identified the human homologue of the MyHC-EO gene in GenBank, and analyzed the 5; upstream region, which revealed a paucity of muscle-specific transcription factor binding sites compared with the other MyHC genes. These features are likely to be critical to the unique regulation and tissue-specific expression of the MyHC-EO/IIL gene. Phylogenetic analysis indicates that MyHC-EO/IIL diverged from an ancestral MyHC gene to generate the first specialized fast myosin. The catalytic S1 head domain is more closely related to the fast MyHCs, while the rod is more closely related to the slow/cardiac MyHCs. The exon boundaries of the MyHC-EO are identical to those of the embryonic MyHC gene and virtually identical to those of the &agr; and (&bgr;) cardiac genes. This implies that most of the current exon boundaries were present in the ancestral gene, predating the duplications that generated the family of skeletal and cardiac myosin genes.

Cloning and characterization of the novel thyroid and eye muscle shared protein G2s: autoantibodies against G2s are closely associated with ophthalmopathy in patients with Graves' hyperthyroidism.

Serum autoantibodies against eye muscle antigens are closely linked with thyroid-associated ophthalmopathy (TAO), although their significance is unclear. The two antigens that are most often recognized are eye muscle membrane proteins with molecular masses of 55 and 64 kDa, as determined from immunoblotting with crude human or porcine eye muscle membranes. We cloned a fragment of the 55-kDa protein by screening an eye muscle expression library with affinity-purified anti-55 kDa protein antibody prepared from a TAO patient's serum. A complementary DNA (cDNA) encoding a novel protein, which we have called G2s, was sequenced on both strands, and its size was 411 bp. The open reading frame of G2s corresponded to a 121-amino acid peptide with a size of 1.4 kb. Using the rapid amplification of 5'-cDNA ends technique we were able to clone an additional 0.3 kb of the protein. G2s did not share significant homologies with any other entered protein in computer databases and had one putative transmembrane domain. Using the 1.4 kb cDNA as probe in Northern blotting of a panel of messenger ribonucleic acids prepared from human tissues, the parent protein was shown to correspond to a large molecule of about 5.8 kb with a calculated molecular mass of approximately 220 kDa, consistent with earlier immunoblot studies performed in the absence of reducing agents. G2s was strongly expressed in eye muscle, thyroid, and other skeletal muscle and to a lesser extent in pancreas, liver, lung, and heart muscle, but not in kidney or orbital fibroblasts. We tested sera from patients with Graves' hyperthyroidism with and without ophthalmopathy and from control patients and subjects for antibodies against a G2s fusion protein by immunoblotting and enzyme-linked immunosorbent assay. In immunoblotting, antibodies reactive with G2s were identified in 70% of patients with TAO of less than 3 yr duration, 53% with TAO of more than 3 yr duration, 36% with Graves' hyperthyroidism without evident ophthalmopathy, 17% with Hashimoto's thyroiditis, 3% with type 1 diabetes, 23% with nonimmunological thyroid disorders, and 16% of normal subjects. The prevalences, compared to normal values, were significant for the two groups of patients with TAO, but not for the other groups. Tests were positive in 54% of patients with active TAO, 33% with chronic ophthalmopathy, 36% with Graves' hyperthyroidism, 54% with Hashimoto's thyroiditis, 23% with type 1 diabetes, and in 11% of normal subjects using enzyme-linked immunosorbent assay. The antibodies predicted the development of the ocular myopathy subtype of TAO in six of seven patients and the congestive ophthalmopathy subtype in seven of eight patients, respectively, with Graves' hyperthyroidism studied prospectively during and after antithyroid drug therapy. Antibodies reactive with G2s may be early markers of ophthalmopathy in patients with Graves' hyperthyroidism. Because G2s is expressed in both thyroid and eye muscle, immunoreactivity against a shared epitope in the two tissues may explain the well known link between thyroid autoimmunity and ophthalmopathy.

Complex three-dimensional patterns of myosin isoform expression: differences between and within specific extraocular muscles.

Because complex structural differences in adult extraocular muscles may have physiological and pathophysiological significance, the three-dimensional pattern of myosin heavy chain (MHC) isoform expression within the orbital and global layers of the muscle bellies compared with the distal tendon ends was quantitatively assessed. Three of the six extraocular muscles of adult rabbits were examined for immunohistologic expression of all fast, fast IIA/X, slow, neonatal and developmental MHC isoforms. The percentages of myofibers positive for each of these 5 myosin isoforms were determined in the orbital and global layers. There were relatively similar patterns of fast and slow MHC expression in the orbital and global layers of each of the three muscles examined. There were high levels of developmental MHC in the orbital layers, but significantly fewer developmental MHC positive myofibers in the global layer. The most variable expression was found with the neonatal MHC. There were significant differences between the longitudinal expression of the various isoforms in the middle of each muscle compared with the tendon end. In the orbital layer of all three muscles examined, the large numbers of fibers positive for fast MHC in the middle of the muscle dramatically decreased at the tendon end, with a concomitant increase in expression of slow myosin. There was a greater number of developmental MHC-positive myofibers at the tendon end than in the middle of the muscle in all three muscles examined. In the global layer, the IIA/X-positive myofibers comprised only half of the total number of fast-positive myofibers whereas in the orbital layer they comprised all or almost all of the fast positive myofibers. The configuration of the extraocular muscles is more complex than might be indicated by previous studies. The lateral rectus muscle had the most individual pattern of MHC expression when compared with the inferior rectus and inferior oblique muscles. Together with dramatic cross-sectional MHC fiber type differences between the orbital and global layers of the muscles, there are pronounced longitudinal differences in the proportions of myofibers expressing these five MHC isoforms in the middle region of the muscles and those in the distal tendon ends. This longitudinal progression appears to occur both within single myofibers, as well as within the series of myofibers that comprise the length of the muscle. We also confirm that the number of myofibers is reduced at the tendonous end while the cross-sectional area of each of the remaining myofibers is proportionally increased with regard to those in the muscle belly. Future studies may yet require two additional schemes for anatomic classification of the named extraocular muscles. One will be based on immunohistochemical features of their constituent myofibers as a supplement to classifications based on their electron microscopic appearance, innervation patterns or relative position with regard to the globe and orbit. Another will be based on the proportional length and longitudinal position of individual myofibers within an individual extraocular muscle.