Extraocular Muscle Fibers Research Articles

Extreme Tolerance of Extraocular Muscles to Diseases and Aging: Why and How?

The extraocular muscles (EOMs) possess unique characteristics that set them apart from other skeletal muscles. These muscles, responsible for eye movements, exhibit remarkable resistance to various muscular dystrophies and aging, presenting a significant contrast to the vulnerability of skeletal muscles to these conditions. In this review, we delve into the cellular and molecular underpinnings of the distinct properties of EOMs. We explore their structural complexity, highlighting differences in fiber types, innervation patterns, and developmental origins. Notably, EOM fibers express a diverse array of myosin heavy-chain isoforms, retaining embryonic forms into adulthood. Moreover, their motor innervation is characterized by a high ratio of nerve fibers to muscle fibers and the presence of unique neuromuscular junctions. These features contribute to the specialized functions of EOMs, including rapid and precise eye movements. Understanding the mechanisms behind the resilience of EOMs to disease and aging may offer insights into potential therapeutic strategies for treating muscular dystrophies and myopathies affecting other skeletal muscles.

Distribution and postnatal development of chondroitin sulfate proteoglycans in the perineuronal nets of cholinergic motoneurons innervating extraocular muscles

Fine control of extraocular muscle fibers derives from two subpopulations of cholinergic motoneurons in the oculomotor-, trochlear- and abducens nuclei. Singly- (SIF) and multiply innervated muscle fibers (MIF) are supplied by the SIF- and MIF motoneurons, respectively, representing different physiological properties and afferentation. SIF motoneurons, as seen in earlier studies, are coated with chondroitin sulfate proteoglycan rich perineuronal nets (PNN), whereas MIF motoneurons lack those. Fine distribution of individual lecticans in the composition of PNNs and adjacent neuropil, as well as the pace of their postnatal accumulation is, however, still unknown. Therefore, the present study aims, by using double immunofluorescent identification and subsequent morphometry, to describe local deposition of lecticans in the perineuronal nets and neuropil of the three eye movement nuclei. In each nucleus PNNs were consequently positive only with WFA and aggrecan reactions, suggesting the dominating role of aggrecan is PNN establishment. Brevican, neurocan and versican however, did not accumulate at all in PNNs but were evenly and moderately present throughout the neuropils. The proportion of PNN bearing motoneurons appeared 76% in oculomotor-, 72.2% in trochlear- and 78.3% in the abducens nucleus. We also identified two morphological subsets of PNNs, the focal and diffuse nets of SIF motoneurons. The process of CSPG accumulation begins just after birth, although considerable PNNs occur at week 1 age around less than half of the motoneurons, which ratio doubles until 2-month age. These findings may be related to the postnatal establishment of the oculokinetic network, performing different repertoires of voluntary eye movements in functionally afoveolate and foveolate animals.

Functional Organization of Extraocular Motoneurons and Eye Muscles

Eye movements are indispensable for visual image stabilization during self-generated and passive head and body motion and for visual orientation. Eye muscles and neuronal control elements are evolutionarily conserved, with novel behavioral repertoires emerging during the evolution of frontal eyes and foveae. The precise execution of eye movements with different dynamics is ensured by morphologically diverse yet complementary sets of extraocular muscle fibers and associated motoneurons. Singly and multiply innervated muscle fibers are controlled by motoneuronal subpopulations with largely selective premotor inputs from task-specific ocular motor control centers. The morphological duality of the neuromuscular interface is matched by complementary biochemical and molecular features that collectively assign different physiological properties to the motor entities. In contrast, the functionality represents a continuum where most motor elements contribute to any type of eye movement, although within preferential dynamic ranges, suggesting that signal transmission and muscle contractions occur within bands of frequency-selective pathways.

Myosin heavy chains in extraocular muscle fibres: Distribution, regulation and function.

This review examines kinetic properties and distribution of the 11 isoforms of myosin heavy chain (MyHC) expressed in extraocular muscle (EOM) fibre types and the regulation and function of these MyHCs. Although recruitment and discharge characteristics of ocular motoneurons during fixation and eye movements are well documented, work directly linking these properties with motor unit contractile speed and MyHC composition is lacking. Recruitment of motor units according to Henneman's size principle has some support in EOMs but needs consolidation. Both neurogenic and myogenic mechanisms regulate MyHC expression as in other muscle allotypes. Developmentally, multiply-innervated (MIFs) and singly-innervated fibres (SIFs) are derived presumably from distinct myoblast lineages, ending up expressing MyHCs in the slow and fast ends of the kinetic spectrum respectively. They modulate the synaptic inputs of their motoneurons through different retrogradely transported neurotrophins, thereby specifying their tonic and phasic impulse patterns. Immunohistochemical analyses of EOMs regenerating in situ and in limb muscle beds suggest that the very impulse patterns driving various ocular movements equip effectors with appropriate MyHC compositions and speeds to accomplish their tasks. These experiments also suggest that satellite cells of SIFs and MIFs are distinct lineages expressing different MyHCs during regeneration. MyHC compositions and functional characteristics of orbital fibres show longitudinal variations that facilitate linear ocular rotation during saccades. Palisade endings on global MIFs are postulated to respond to active and passive tensions by triggering axon reflexes that play important roles during fixation, saccades and vergence. How EOMs implement Listings law during ocular rotation is discussed.

EOM Pulleys and Sequelae: A Critical Review.

The discovery of extraocular muscle (EOM) pulleys resolved long-standing issues in oculomotor physiology, revived interest in EOM function generally, and led to several new theories. We describe the pulley concept of Miller and Demer (M-D Pulleys) and briefly review evidence, distinguishing this well-supported notion from the Active Pulley Hypothesis (APH) and the EOM Compartments hypothesis, and critically reviewing the methodologies and evidence on which the latter are based. We analyze evidence on mechanical independence of individual EOM fibers, implications of nerve tracing for functional independence of EOM layers and compartments, validity of image-based methods of assessing EOM contraction, and data analysis issues. M-D Pulleys are well-supported by several lines of evidence from several labs. The APH, which predicts relative movements of EOM lamina sufficient to alter muscle actions, has been effectively disproved. The width-wise articulations of EOM Compartments, in contrast, might produce significant contractile oculorotary force gradients across muscle tendons, although existing evidence is unconvincing. We suggest how this hypothesis could be effectively tested.

Ultrastructural changes of extraocular muscles in strabismus patients

ABSTRACTStrabismus is an ocular disorder characterized by partial or complete inability to keep eye alignment. It represents a very common ocular problem at ophthalmology clinics worldwide. The current study aimed to show the most encountered ultrastructural changes in extraocular muscles (EOMs) collected from patients with different forms of strabismus. Nine specimens of EOMs were collected from five patients during strabismus correction surgery and processed for light and electron microscopy examinations. Histologically, skeletal muscle fibers in normal EOMs appeared tight and normally arranged with clear striations. In strabismic muscles, the fibers appeared disarranged, and atrophied, swollen and disintegrated in some situations. By transmission electron microscopy, normal EOMs were formed of skeletal muscle fibers with intact basal membrane and sarcolemma, tightly aligned myofibrils with well-arranged sarcomeres, Z line and H zone, and normally distributed mitochondria. On the other hand, strabismic EOMs revealed vacuolation and degeneration of myofibrils, accumulation of lipid droplets, subsarcolemmal inclusions and clustering of mitochondria. EOMs obtained from a Down syndrome patient with V-pattern infantile esotropia showed extensive vacuolation and disintegration of myofibrils, and extra- and intracellular deposition of collagen fibers. Interestingly, some skeletal muscle cells exhibited features of autophagic cell death with a trial of engulfing process by neighboring cells. In conclusion, our study traces some characteristic ultrastructural changes in strabismic EOMs, most notably, extensive vacuolation, clustering of mitochondria, degeneration of myofibrils and autophagic changes. These changes might be emphasized as possibly secondary to strabismus.

An experimental study on the induction of Graves′ hyperthyroidism with Graves′ ophthalmopathy in BALB/c mice by TSHR gene immunization

Objective To construct a model of Graves′ disease(GD) with (or) Graves′ ophthalmopathy(GO) in BALB/c mice by immunization with pcDNA3.1/TSHR289. Methods pcDNA3.1/TSHR289 was injected into the bilateral gastrocnemius muscle of 35 model mice and electroporation was immediately performed. 10 control mice were injected with sterile saline and electroporated, while 5 blank mice were injected with sterile saline only. Each group of mice was immunized at 1, 4, 7, and 10 weeks, respectively. Serum total T4, TSH, TSAb, and TSBAb were measured before immunization, 2 weeks after each immunization, as well as 5 and 8 weeks after the last immunization. CT scan was used to evaluate the morphological changes of the eyes of the mice.99mTcO4- imaging was used to measure the thyroid uptake function, and the pathological changes of the thyroid and orbital tissues were evaluated by HE staining. Results After the 2nd time immunization, the serum concentrations of TT4, TSAb and TSBAb in GD mice were significantly higher than those of control and blank groups(F=13.781, 31.435, 36.112, P<0.01, respectively). The TSH continued to be significantly lower than that of control and blank groups(F=13.966, P<0.01). After the 4th time immunizations, the ability of uptaking99mTcO4- in GD mice thyroid was significantly enhanced compared with the control group. The thyroid goiter with a large amount of lymphocyte infiltration, and the thyroid follicle was thin. CT scan of GO mice showed thickening and swelling of the extraocular muscles, and no abnormalities in tendon and muscle attachment points. HE staining showed thickening of extraocular muscle fibers, lymphocyte infiltration of extraocular muscles and orbital tissue, increased hyaluronic acid, and infiltration of fat cells. Conclusion GD or GO model can be successfully induced by multiple intramuscular injection of pcDNA3.1/TSHR289 in BALB/c mice. Key words: Graves′ disease; Graves ophthalmopathy; TSHR; Mice models; Electroporation

Distinct segregation of the pathogenic m.5667G>A mitochondrial tRNAAsn mutation in extraocular and skeletal muscle in chronic progressive external ophthalmoplegia

Chronic progressive external ophthalmoplegia (CPEO) is a frequent clinical manifestation of disorders caused by pathogenic mitochondrial DNA mutations. However, for diagnostic purposes skeletal muscle tissue is used, since extraocular muscle tissue is usually not available for work-up. In the present study we aimed to identify causative factors that are responsible for extraocular muscle to be primarily affected in CPEO. We performed comparative histochemical and molecular genetic analyses of extraocular muscle and skeletal muscle single fibers in a case of isolated CPEO caused by the heteroplasmic m.5667G>A mutation in the mitochondrial tRNAAsn gene (MT-TN). Histochemical analyses revealed higher proportion of cytochrome c oxidase deficient fibers in extraocular muscle (41%) compared to skeletal muscle (10%). However, genetic analyses of single fibers revealed no significant difference either in the mutation loads between extraocular muscle and skeletal muscle cytochrome c oxidase deficient single fibers (extraocular muscle 86% ± 4.6%; skeletal muscle 87.8 %± 5.7%, p = 0.246) nor in the mutation threshold (extraocular muscle 74% ± 3%; skeletal muscle 74% ± 4%). We hypothesize that higher proportion of cytochrome c oxidase deficient fibers in extraocular muscle compared to skeletal muscle might be due to facilitated segregation of the m.5667G>A mutation into extraocular muscle, which may explain the preferential ocular manifestation and clinically isolated CPEO.

Passive eye movements induced by electromagnetic force (EMF) in rats.

Accurate information on eye position in the orbit is available from visual feedback, efference copy of the oculomotor commands and proprioceptive signals from the extraocular muscles (EOM). Whereas visual feedback and oculomotor commands have been extensively studied, central processing of EOM proprioceptive signals remains to be elucidated. A challenge to the field is to develop an approach to induce passive eye movements without physically contacting the eyes. A novel method was developed to generate passive eye movements in rats. A small rare-earth magnet disk (0.7 mm diameter, 0.5 mm thickness) was attached to the surface of a rat’s eyeball. A metal rod (5 mm diameter) wrapped with an electromagnetic (EM) coil was placed near the magnet (8–15 mm). By passing currents to the EM coil, electromagnetic force (EMF) was generated and acted upon the magnet and induced passive eye movements. The EMF induced well-defined passive eye movements, whose directions were dependent on current polarity and amplitudes and peak velocities were dependent on current intensity and duration. Peak velocities of the EMF-induced eye movements were linearly related to amplitudes, exhibiting main sequence relationships similar to that of saccades in awake rats and eye movements induced by electrical microstimulation of the abducens nucleus in anesthetized rats. Histological examination showed that repetitive EMF stimulations did not appear to result in damages in the EOM fibers. These results validated the EMF approach as a novel tool to investigate EOM proprioceptive signals and their roles in visual localization and gaze control.

MITOCHONDRIAL DISEASES (Posters): P.191Single muscle fiber analysis of extraocular and skeletal muscles in a CPEO patient harboring a pathogenic point mutation in the MT-TN gene

Chronic progressive external ophthalmoplegia (CPEO) is a frequent feature of mitochondrial disorders and usually associated with mitochondrial DNA (mtDNA) mutations. Skeletal muscle (SKM) tissue is most frequently used for biochemical analyses and mitochondrial genome testing. However, extraocular muscle (EOM) is the clinically most affected tissue but usually not available for routine work-up. Consequently, systematic data on EOM is limited and the reason for preferential clinical affection remains unclear. We addressed this unsolved question by histochemical and genetic analyses of EOM and SKM single muscle fibers in a patient with isolated CPEO caused by a heteroplasmic point mutation in the MT-TN gene. The histochemical analysis showed higher absolute numbers of cytochrome c oxidase (COX)-deficient EOM fibers compared to COX-deficient SKM fibers. However, genetic analyses by restriction fragment length polymorphism revealed no significant difference in the mutation loads between COX-negative single muscle fibers in EOM compared to SKM. Quantitative single fiber real-time PCR revealed higher mtDNA copy numbers in single muscle fibers of EOM compared to SKM. COX-negative single muscle fibers of EOM and SKM showed significantly higher mtDNA copy numbers compared to COX-positive fibers suggestive of a compensatory mtDNA proliferation. We show that high loads of the MT-TN mutation correlate with a biochemical loss of COX activity in single muscle fibers of EOM and SKM at a similar threshold. The higher absolute numbers of COX-negative fibers in EOM compared to SKM might be caused by facilitated segregation of the mutation into the EOM providing thereby a possible explanation of the preferential ocular manifestation in CPEO.

Changes in Myosin Heavy Chain Isoforms Along the Length of Orbital Fibers in Rabbit Extraocular Muscle.

Extraocular muscles express 10 myosin heavy chain (MyHC) isoforms that cater for a wide range of contractile speeds. We aim to characterize the variations in MyHC expression along the length of singly (SIFs) and multiply innervated fibers (MIFs) in the orbital layer of rabbit superior rectus muscle. Monospecific antibodies to nine MyHCs, including an anti-slow-tonic antibody characterized here were used to immunohistochemically map variations in MyHC distribution in serial sections along the muscle's full length. The fastest MyHC, EO, is expressed at the endplate zone (EPZ) of SIFs, flanked proximally and distally by segments expressing the slower 2A, with or without embryonic MyHC. MIFs with constant diameter express α-cardiac MyHC at the EPZ, flanked by segments co-expressing α-cardiac/embryonic and possibly slow-tonic MyHCs. MIFs with varying diameter also express α-cardiac MyHC at the EPZ in their thin, central region, flanked by thin segments co-expressing α-cardiac/embryonic MyHCs, with long proximal and distal extensions of larger diameter that co-express embryonic/slow-tonic and α-cardiac or β/slow MyHCs. Orbital fiber types express multiple MyHCs, with faster ones in SIFs, slower ones in MIFs, but all have fast EPZs and slower end segments. We hypothesize that these unique MyHC distributions enable these fibers to relax in two kinetically distinct phases while acting in an antagonistic manner during a saccade: the fast phases facilitate acceleration of eyeball rotation during agonist contraction, while the slow phases help its deceleration toward the visual target, thereby linearizing the saccade. These properties also facilitate pulley movements to implement Listing's law.

A central mesencephalic reticular formation projection to medial rectus motoneurons supplying singly and multiply innervated extraocular muscle fibers.

We recently demonstrated a bilateral projection to the supraoculomotor area from the central mesencephalic reticular formation (cMRF), a region implicated in horizontal gaze changes. C-group motoneurons, which supply multiply innervated fibers in the medial rectus muscle, are located within the primate supraoculomotor area, but their inputs and function are poorly understood. Here, we tested whether C-group motoneurons in Macaca fascicularis monkeys receive a direct cMRF input by injecting this portion of the reticular formation with anterograde tracers in combination with injection of retrograde tracer into the medial rectus muscle. The results indicate that the cMRF provides a dense, bilateral projection to the region of the medial rectus C-group motoneurons. Numerous close associations between labeled terminals and each multiply innervated fiber motoneuron were present. Within the oculomotor nucleus, a much sparser ipsilateral projection onto some of the A- and B- group medial rectus motoneurons that supply singly innervated fibers was observed. Ultrastructural analysis demonstrated a direct synaptic linkage between anterogradely labeled reticular terminals and retrogradely labeled medial rectus motoneurons in all three groups. These findings reinforce the notion that the cMRF is a critical hub for oculomotility by proving that it contains premotor neurons supplying horizontal extraocular muscle motoneurons. The differences between the cMRF input patterns for C-group versus A- and B-group motoneurons suggest the C-group motoneurons serve a different oculomotor role than the others. The similar patterns of cMRF input to C-group motoneurons and preganglionic Edinger-Westphal motoneurons suggest that medial rectus C-group motoneurons may play a role in accommodation-related vergence.

Treatment of convergence insufficiency exotropia in adults using a selective muscle fiber surgery treatment algorithm

Treatment of adults with convergence insufficiency (CI) exotropia is challenging as surgery frequently results in distance and lateral gaze esotropia. Recent evidence demonstrates differential compartmental function of extraocular muscle fiber groups. Selective surgery of muscle fiber groups may diminish distance–near disparities (NDD) while maintaining alignment at distance.

Aquaporin expression and localization in the rabbit eye

Aquaporins (AQPs) are important for ocular homeostasis and function. While AQP expression has been investigated in ocular tissues of human, mouse, rat and dog, comprehensive data in rabbits are missing. As rabbits are frequently used model organisms in ophthalmic research, the aim of this study was to analyze mRNA expression and to localize AQPs in the rabbit eye. The results were compared with the data published for other species. In cross sections of New Zealand White rabbit eyes AQP0 to AQP5 were labeled by immunohistology and analyzed by confocal microscopy. Immunohistological findings were compared to mRNA expression levels, which were analyzed by quantitative reverse transcription real time polymerase chain reaction (qRT-PCR). The primers used were homologous against conserved regions of AQPs. In the rabbit eye, AQP0 protein expression was restricted to the lens, while AQP1 was present in the cornea, the chamber angle, the iris, the ciliary body, the retina and, to a lower extent, in optic nerve vessels. AQP3 and AQP5 showed immunopositivity in the cornea. AQP3 was also present in the conjunctiva, which could not be confirmed for AQP5. However, at a low level AQP5 was also traceable in the lens. AQP4 protein was detected in the ciliary non-pigmented epithelium (NPE), the retina, optic nerve astrocytes and extraocular muscle fibers. For most tissues the qRT-PCR data confirmed the immunohistology results and vice versa. Although species differences exist, the AQP protein expression pattern in the rabbit eye shows that, especially in the anterior section, the AQP distribution is very similar to human, mouse, rat and dog. Depending on the ocular regions investigated in rabbit, different protein and mRNA expression results were obtained. This might be caused by complex gene regulatory mechanisms, post-translational protein modifications or technical limitations. However, in conclusion the data suggest that the rabbit is a useful in-vivo model to study AQP function and the effects of direct and indirect intervention strategies to investigate e. g. mechanisms for intraocular pressure modulation or cornea transparency regulation.

Compartmental Innervation of the Superior Oblique Muscle in Mammals.

Intramuscular innervation of mammalian horizontal rectus extraocular muscles (EOMs) is compartmental. We sought evidence of similar compartmental innervation of the superior oblique (SO) muscle. Three fresh bovine orbits and one human orbit were dissected to trace continuity of SO muscle and tendon fibers to the scleral insertions. Whole orbits were also obtained from four humans (two adults, a 17-month-old child, and a 33-week stillborn fetus), two rhesus monkeys, one rabbit, and one cow. Orbits were formalin fixed, embedded whole in paraffin, serially sectioned in the coronal plane at 10-μm thickness, and stained with Masson trichrome. Extraocular muscle fibers and branches of the trochlear nerve (CN4) were traced in serial sections and reconstructed in three dimensions. In the human, the lateral SO belly is in continuity with tendon fibers inserting more posteriorly on the sclera for infraducting mechanical advantage, while the medial belly is continuous with anteriorly inserting fibers having mechanical advantage for incycloduction. Fibers in the monkey superior SO insert more posteriorly on the sclera to favor infraduction, while the inferior portion inserts more anteriorly to favor incycloduction. In all species, CN4 bifurcates prior to penetrating the SO belly. Each branch innervates a nonoverlapping compartment of EOM fibers, consisting of medial and lateral compartments in humans and monkeys, and superior and inferior compartments in cows and rabbits. The SO muscle of humans and other mammals is compartmentally innervated in a manner that could permit separate CN4 branches to selectively influence vertical versus torsional action.

Compartmentalization of extraocular muscle function.

Ocular motor diversity exceeds capabilities of only six extraocular muscles (EOMs), but this deficiency is overcome by the plethora of fibers within individual EOMs surpassing requirements of homogeneous actuators. This paper reviews emerging evidence that regions of individual EOMs can be differentially innervated to exert independent oculorotary torques, broadening the oculomotor repertoire, and potentially explaining diverse strabismus pathophysiology. Parallel structure characterizes EOM and tendon fibers, with little transverse coupling of experimentally imposed or actively generated tension. This arrangement enables arbitrary groupings of tendon and muscle fibers to act relatively independently. Coordinated force generation among EOM fibers occurs only upon potentially mutable coordination of innervational commands, whose central basis is suggested by preliminary findings of apparent compartmental segregation of abducens motor neuron pools. Humans, monkeys, and other mammals demonstrate separate, nonoverlapping intramuscular nerve arborizations in the superior vs inferior compartments of the medial rectus (MR) and lateral rectus (LR) EOMs that could apply force at the superior vs inferior portions of scleral insertions, and in the medial vs lateral compartments of the superior oblique that act at the equatorial vs posterior scleral insertions that might preferentially implement incycloduction vs infraduction. Magnetic resonance imaging of the MR during several physiological ocular motor behaviors indicates differential compartmental function. Differential compartmental pathology can influence clinical strabismus. Partial abducens palsy commonly affects the superior LR compartment more than the inferior, inducing vertical strabismus that might erroneously be attributed to cyclovertical EOM pathology. Surgery may selectively manipulate EOM compartments.

Intermediate filaments in the human extraocular muscles.

To investigate the distribution of the intermediate filament (IF) proteins desmin, vimentin, and nestin in human extraocular muscles (EOMs). Healthy adult EOM samples were serially sectioned (5 and 1 μm) and processed for immunohistochemistry, with specific antibodies (Abs) against desmin, vimentin, and nestin and different myosin heavy chains (MyHCs), including the newly characterized Ab MYH7b against MyHC slow tonic. The distribution of desmin was also studied in EOMs at 16 to 18 weeks of gestation. Desmin was present in the vast majority of muscle fibers. Notably, muscle fibers that contained MyHC slow tonic were either unlabeled or very weakly labeled with three different Abs against desmin. These muscle fibers had normal cytoarchitecture and intact basement membrane. In fetal muscle, desmin was also absent or weak in myotubes containing MyHC slow tonic. Nestin was detected in a large proportion of muscle fibers in the orbital layer and to some extent also in the global layer, whereas no muscle fibers contained vimentin. Desmin and nestin were enriched at neuromuscular junctions, as in limb muscle. In contrast, some myotendinous junctions lacked desmin or nestin. The human EOMs differed significantly from the other muscles in the body with respect to their IF composition. Desmin, hitherto regarded as a ubiquitous muscle cytoskeletal protein, was absent or only present in trace amounts in a subset of normal muscle fibers in adult and fetal EOMs. Nestin, normally downregulated early in the postnatal period, was present in a high proportion of adult muscle fibers.

Complex tropomyosin and troponin T isoform expression patterns in orbital and global fibers of adult dog and rat extraocular muscles

We reported marked differences in the myosin heavy and light chain (MHC and MLC) isoform composition of fast and slow fibers between the global and orbital layers of dog extraocular muscles. Many dog extraocular fibers, especially orbital fibers, have MHC and MLC isoform patterns that are distinct from those in limb skeletal muscles. Additional observations suggested possible differences in the tropomyosin (Tm) and troponin T (TnT) isoform composition of global and orbital fibers. Therefore, we tested, using SDS-PAGE and immunoblotting, whether differences in Tm and TnT isoform expression do, in fact, exist between global and orbital layers of dog and rat EOMs and to compare expression patterns among identified fast and slow single fibers from both muscle layers. The Tm isoforms expressed in global fast and slow fibers are the same as in limb fast (α-Tm and β-Tm) and slow (γ-Tm and β-Tm) fibers, respectively. Orbital slow orbital fibers, on the other hand, each co-express all three sarcomeric Tm isoforms (α, β and γ). The results indicate that fast global and orbital fibers express only fast isoforms of TnT, but the relative amounts of the individual isoforms are different from those in limb fast muscle fibers and an abundant fast TnT isoform in the orbital layer was not detected in fast limb muscles. Slow fibers in both layers express slow TnT isoforms and the relative amounts also differ from those in limb slow fibers. Unexpectedly, significant amounts of cardiac TnT isoforms were also detected in slow fibers, especially in the orbital layer in both species. TnI and TnC isoform patterns are the same as in fast and slow fibers in limb muscles. These results expand the understanding of the elaborate diversity in contractile protein isoform expression in mammalian extraocular muscle fibers and suggest that major differences in calcium-activation properties exist among these fibers, based upon Tm and TnT isoform expression patterns.

Distinct Changes in Synaptic Protein Composition at Neuromuscular Junctions of Extraocular Muscles versus Limb Muscles of ALS Donors

The pathophysiology of amyotrophic lateral sclerosis (ALS) is very complex and still rather elusive but in recent years evidence of early involvement of the neuromuscular junctions (NMJs) has accumulated. We have recently reported that the human extraocular muscles (EOMs) are far less affected than limb muscles at the end-stage of ALS from the same donor. The present study aimed to compare the differences in synaptic protein composition at NMJ and in nerve fibers between EOM and limb muscles from ALS donors and controls. Neurofilament light subunit and synaptophysin decreased significantly at NMJs and in nerve fibers in limb muscles with ALS whereas they were maintained in ALS EOMs. S100B was significantly decreased at NMJs and in nerve fibers in both EOMs and limb muscles of ALS donors, but other markers confirmed the presence of terminal Schwann cells in these NMJs. p75 neurotrophin receptor was present in nerve fibers but absent at NMJs in ALS limb muscles. The EOMs were able to maintain the integrity of their NMJs to a very large extent until the end-stage of ALS, in contrast to the limb muscles. Changes in Ca2+ homeostasis, reflected by altered S100B distribution, might be involved in the breakdown of nerve-muscle contact at NMJs in ALS.

Effects of sequential injections of hepatocyte growth factor and insulin-like growth factor-I on adult rabbit extraocular muscle

To determine whether hepatocyte growth factor (HGF) and insulin-like growth factor-I (IGF-I) have synergistic effects in promoting extraocular muscle fiber growth and force generation. A superior rectus muscle of adult rabbits was treated with either a single injection of HGF or sequential injections of HGF followed 1 week later by IGF-I. One week after HGF alone and 1 week after the IGF-I injection, the superior rectus muscles from treated and control orbits were examined for alterations in force generation as well as changes in myofiber size. Injection of HGF alone did not result in changes to muscle force, specific tension, or myofiber cross-sectional area; however, it did result in a significant increase in numbers of satellite cells. Sequential injection of HGF and IGF-I resulted in significantly increased force, specific tension, and myofiber cross-sectional areas as well as increased numbers of satellite cells. Preinjection with HGF augments the treatment effect of IGF-I. This synergistic effect is likely a result of HGF-induced activation of satellite cells and should allow a reduction in IGF-I dosing required to produce a given increase in extraocular muscle force generation.