Extraocular muscle stem cells exhibit distinct cellular properties associated with non-muscle molecular signatures.

Skeletal muscle stem cells (MuSCs) are recognised as functionally heterogeneous. Cranial MuSCs are reported to have greater proliferative and regenerative capacity when compared with those in the limb. A comprehensive understanding of the mechanisms underlying this functional heterogeneity is lacking. Here, we have used clonal analysis, live imaging and single cell transcriptomic analysis to identify crucial features that distinguish extraocular muscle (EOM) from limb muscle stem cell populations. A MyogeninntdTom reporter showed that the increased proliferation capacity of EOM MuSCs correlates with deferred differentiation and lower expression of the myogenic commitment gene Myod. Unexpectedly, EOM MuSCs activated in vitro expressed a large array of extracellular matrix components typical of mesenchymal non-muscle cells. Computational analysis underscored a distinct co-regulatory module, which is absent in limb MuSCs, as driver of these features. The EOM transcription factor network, with Foxc1 as key player, appears to be hardwired to EOM identity as it persists during growth, disease and in vitro after several passages. Our findings shed light on how high-performing MuSCs regulate myogenic commitment by remodelling their local environment and adopting properties not generally associated with myogenic cells.

Interplay between Pitx2 and Pax7 temporally governs specification of extraocular muscle stem cells.

Gene regulatory networks that act upstream of skeletal muscle fate determinants are distinct in different anatomical locations. Despite recent efforts, a clear understanding of the cascade of events underlying the emergence and maintenance of the stem cell pool in specific muscle groups remains unresolved and debated. Here, we invalidated Pitx2 with multiple Cre-driver mice prenatally, postnatally, and during lineage progression. We showed that this gene becomes progressively dispensable for specification and maintenance of the muscle stem (MuSC) cell pool in extraocular muscles (EOMs) despite being, together with Myf5, a major upstream regulator during early development. Moreover, constitutive inactivation of Pax7 postnatally led to a greater loss of MuSCs in the EOMs compared to the limb. Thus, we propose a relay between Pitx2, Myf5 and Pax7 for EOM stem cell maintenance. We demonstrate also that MuSCs in the EOMs adopt a quiescent state earlier that those in limb muscles and do not spontaneously proliferate in the adult, yet EOMs have a significantly higher content of Pax7+ MuSCs per area pre- and post-natally. Finally, while limb MuSCs proliferate in the mdx mouse model for Duchenne muscular dystrophy, significantly less MuSCs were present in the EOMs of the mdx mouse model compared to controls, and they were not proliferative. Overall, our study provides a comprehensive in vivo characterisation of MuSC heterogeneity along the body axis and brings further insights into the unusual sparing of EOMs during muscular dystrophy.

MYH13, a superfast myosin expressed in extraocular, laryngeal and syringeal muscles.

MYH13 is a unique type of sarcomeric myosin heavy chain (MYH) first detected in mammalian extraocular (EO) muscles and later also in vocal muscles, including laryngeal muscles of some mammals and syringeal muscles of songbirds. All these muscles are specialized in generating very fast contractions while producing relatively low force, a design appropriate for muscles acting against a much lower load than most skeletal muscles inserting into the skeleton. The definition of the physiological properties of muscle fibres containing MYH13 has been complicated by the mixed fibre type composition of EO muscles and the coexistence of different MYH types within the same fibre. A major advance in this area came from studies on isolated recombinant myosin motors and the demonstration that the affinity of actin-bound human MYH13 for ADP is much weaker than those of fast-type MYH1 (type 2X) and MYH2 (type 2A). This property is consistent with a very fast detachment of myosin from actin, a major determinant of shortening velocity. The MYH13 gene arose early during vertebrate evolution but was characterized only in mammals and birds and appears to have been lost in some teleost fish. The MYH13 gene is located at the 3' end of the mammalian fast/developmental gene cluster and in a similar position to the orthologous cluster in syntenic regions of the songbird genome. MYH13 gene regulation is controlled by a super-enhancer in the mammalian locus and deletion of the neighbouring fast MYH1 and MYH4 genes leads to abnormal MYH13 expression in mouse leg muscles.

Developmental, Physiological and Phylogenetic Perspectives on the Expression and Regulation of Myosin Heavy Chains in Craniofacial Muscles.

This review deals with the developmental origins of extraocular, jaw and laryngeal muscles, the expression, regulation and functional significance of sarcomeric myosin heavy chains (MyHCs) that they express and changes in MyHC expression during phylogeny. Myogenic progenitors from the mesoderm in the prechordal plate and branchial arches specify craniofacial muscle allotypes with different repertoires for MyHC expression. To cope with very complex eye movements, extraocular muscles (EOMs) express 11 MyHCs, ranging from the superfast extraocular MyHC to the slowest, non-muscle MyHC IIB (nmMyH IIB). They have distinct global and orbital layers, singly- and multiply-innervated fibres, longitudinal MyHC variations, and palisade endings that mediate axon reflexes. Jaw-closing muscles express the high-force masticatory MyHC and cardiac or limb MyHCs depending on the appropriateness for the acquisition and mastication of food. Laryngeal muscles express extraocular and limb muscle MyHCs but shift toward expressing slower MyHCs in large animals. During postnatal development, MyHC expression of craniofacial muscles is subject to neural and hormonal modulation. The primary and secondary myotubes of developing EOMs are postulated to induce, via different retrogradely transported neurotrophins, the rich diversity of neural impulse patterns that regulate the specific MyHCs that they express. Thyroid hormone shifts MyHC 2A toward 2B in jaw muscles, laryngeal muscles and possibly extraocular muscles. This review highlights the fact that the pattern of myosin expression in mammalian craniofacial muscles is principally influenced by the complex interplay of cell lineages, neural impulse patterns, thyroid and other hormones, functional demands and body mass. In these respects, craniofacial muscles are similar to limb muscles, but they differ radically in the types of cell lineage and the nature of their functional demands.

Identification and characterization of differentially expressed circular RNAs in extraocular muscle of oculomotor nerve palsy.

BACKGROUND: Oculomotor nerve palsy (ONP) is a neuroparalytic disorder resulting in dysfunction of innervating extraocular muscles (EOMs), of which the pathological characteristics remain underexplored. METHODS: In this study, medial rectus muscle tissue samples from four ONP patients and four constant exotropia (CXT) patients were collected for RNA sequencing. Differentially expressed circular RNAs (circRNAs) were identified and included in functional enrichment analysis, followed by interaction analysis with microRNAs and mRNAs as well as RNA binding proteins. Furthermore, RT-qPCR was used to validate the expression level of the differentially expressed circRNAs. RESULTS: A total of 84 differentially expressed circRNAs were identified from 10,504 predicted circRNAs. Functional enrichment analysis indicated that the differentially expressed circRNAs significantly correlated with skeletal muscle contraction. In addition, interaction analyses showed that up-regulated circRNA_03628 was significantly interacted with RNA binding protein AGO2 and EIF4A3 as well as microRNA hsa-miR-188-5p and hsa-miR-4529-5p. The up-regulation of circRNA_03628 was validated by RT-qPCR, followed by further elaboration of the expression, location and clinical significance of circRNA_03628 in EOMs of ONP. CONCLUSIONS: Our study may shed light on the role of differentially expressed circRNAs, especially circRNA_03628, in the pathological changes of EOMs in ONP.

Effects of myostatin gene knockout on porcine extraocular muscles.

Myostatin (MSTN), a negative regulator of skeletal muscle mass, is not well known in extraocular muscles (EOMs). EOMs are specialized skeletal muscles. Hence, in this study, the effect of MSTN on the superior rectus (SR) and superior oblique (SO) of 2-month-old MSTN knockout (MSTN-/-) and wild-type (WT) pigs of the same genotype was investigated. SR (P < 0.01) and SO (P < 0.001) fiber cross-sectional areas of MSTN-/- pigs were significantly larger than those of WT pigs. Compared with WT pigs, MSTN-/- SO displayed a decrease in type I fibers (WT: 27.24%, MSTN-/-: 10.32%, P < 0.001). Type IIb fibers were higher in MSTN-/- pigs than in WT pigs (WT: 30.38%, MSTN-/-: 62.24%, P < 0.001). The trend in SR was the same as that in SO, although the trend in SO was greater than that in SR. The expression of myogenic differentiation factor (MyoD) and myogenic (MyoG) showed a significant increase in MSTN-/- SO (about 2.5-fold and 2-fold, respectively at the gene expression level, about 1.5-fold at the protein level) compared with WT pigs. MSTN plays an important role in the development of EOMs and regulates the muscle fiber type by modulating the gene expression of MyoD and MyoG in pigs.

Oculomotor nerve guidance and terminal branching requires interactions with differentiating extraocular muscles.

Muscle function is dependent on innervation by the correct motor nerves. Motor nerves are composed of motor axons which extend through peripheral tissues as a compact bundle, then diverge to create terminal nerve branches to specific muscle targets. As motor nerves approach their targets, they undergo a transition where the fasciculated nerve halts further growth then after a pause, the nerve later initiates branching to muscles. This transition point is potentially an intermediate target or guidepost to present specific cellular and molecular signals for navigation. Here we describe the navigation of the oculomotor nerve and its association with developing muscles in mouse embryos. We found that the oculomotor nerve initially grew to the eye three days prior to the appearance of any extraocular muscles. The oculomotor axons spread to form a plexus within a mass of cells, which included precursors of extraocular muscles and other orbital tissues and expressed the transcription factor Pitx2. The nerve growth paused in the plexus for more than two days, persisting during primary extraocular myogenesis, with a subsequent phase in which the nerve branched out to specific muscles. To test the functional significance of the nerve contact with Pitx2+ cells in the plexus, we used two strategies to genetically ablate Pitx2+ cells or muscle precursors early in nerve development. The first strategy used Myf5-Cre-mediated expression of diphtheria toxin A to ablate muscle precursors, leading to loss of extraocular muscles. The oculomotor axons navigated to the eye to form the main nerve, but subsequently largely failed to initiate terminal branches. The second strategy studied Pitx2 homozygous mutants, which have early apoptosis of Pitx2-expressing precursor cells, including precursors for extraocular muscles and other orbital tissues. Oculomotor nerve fibers also grew to the eye, but failed to stop to form the plexus, instead grew long ectopic projections. These results show that neither Pitx2 function nor Myf5-expressing cells are required for oculomotor nerve navigation to the eye. However, Pitx2 function is required for oculomotor axons to pause growth in the plexus, while Myf5-expressing cells are required for terminal branch initiation.

Molecular basis of impaired extraocular muscle function in a mouse model of congenital myopathy due to compound heterozygous Ryr1 mutations.

Mutations in the RYR1 gene are the most common cause of human congenital myopathies, and patients with recessive mutations are severely affected and often display ptosis and/or ophthalmoplegia. In order to gain insight into the mechanism leading to extraocular muscle (EOM) involvement, we investigated the biochemical, structural and physiological properties of eye muscles from mouse models we created knocked-in for Ryr1 mutations. Ex vivo force production in EOMs from compound heterozygous RyR1p.Q1970fsX16+p.A4329D mutant mice was significantly reduced compared with that observed in wild-type, single heterozygous mutant carriers or homozygous RyR1p.A4329D mice. The decrease in muscle force was also accompanied by approximately a 40% reduction in RyR1 protein content, a decrease in electrically evoked calcium transients, disorganization of the muscle ultrastructure and a decrease in the number of calcium release units. Unexpectedly, the superfast and ocular-muscle-specific myosin heavy chain-EO isoform was almost undetectable in RyR1p.Q1970fsX16+p.A4329D mutant mice. The results of this study show for the first time that the EOM phenotype caused by the RyR1p.Q1970fsX16+p.A4329D compound heterozygous Ryr1 mutations is complex and due to a combination of modifications including a direct effect on the macromolecular complex involved in calcium release and indirect effects on the expression of myosin heavy chain isoforms.

Flavonoids Sophoranone Promotes Differentiation of C2C12 and Extraocular Muscle Satellite Cells.

INTRODUCTION: Paralytic strabismus involves a functional loss of extraocular muscles resulting from muscular or neuronal disorders. Currently, only a limited number of drugs are available for functional repair of extraocular muscles. Here, we investigated the effects of a novel drug, flavonoids sophoranone, on the differentiation of extraocular muscles as assessed in bothin vivo and in vitro models. MATERIALS AND METHODS: The effect of flavonoids sophoranone on C2C12 cells was examinedin vitro as evaluated with use of apoptosis, reactive oxygen species (ROS), and cell viability assays. Then, both in vivo and in vitro effects of this drug were examined on the differentiation of C2C12 and satellite cells within extraocular muscles in rabbits. For these latter experiments, RT-PCR and Western blot assays were used to determine expression levels of markers for myogenic differentiation. RESULTS: With use of flavonoids sophoranone concentrations ranging from 0 to 10 µM, no effects were observed upon cell apoptosis, ROS, and cell cycle in C2C12 cells. Based on MTT assay results, flavonoids sophoranone was shown to increase C2C12 cell proliferation. Moreover, flavonoids sophoranone promoted the differentiation of C2C12 and satellite cells within extraocular muscles in rabbits, which were verified as based on cell morphology and expression levels of mRNA and protein markers of myogenic differentiation. Finally, flavonoids sophoranone treatment also increased gene expressions of Myh3, Myog, and MCK. CONCLUSION: The capacity for flavonoids sophoranone to upgrade the differentiation of both C2C12 and satellite cells within extraocular muscles in rabbits at concentrations producing no adverse effects suggest that this drug may provide a safe and effective means to promote repair of damaged extraocular muscles.

Integrative transcriptomics and proteomic analysis of extraocular muscles from patients with thyroid-associated ophthalmopathy.

Our study aimed to reveal the underlying pathologic mechanisms of thyroid-associated ophthalmopathy (TAO) by integrative transcriptomics and proteomic analysis of extraocular muscles (EOM). The study involved 11 TAO patients (clinical activity score ≤ 2) and 11 control donors. Total RNA was extracted from EOM samples of 5 TAO patients and 5 control individuals for gene microarray analysis to reveal differentially expressed genes. Concurrently, EOM samples from 3 TAO patients and 3 control individuals were lysed for quantitative proteomic analysis. Differentially expressed genes and proteins were identified, followed by functional and pathway enrichment analysis and protein-protein interaction network construction. Concordance between proteins and transcripts was examined, and functional annotations were conducted. Expressions of versican (VCAN) and lipocalin 1 (LCN1) in EOM samples from another 3 TAO patients and 3 control individuals were measured by western blotting. In total, 952 genes and 137 proteins were identified as differentially expressed, as well as 96 differentially expressed proteins without significantly changed mRNA abundance. Proteins mainly related to the composition (such as MYH1, MYH2, and MYH13) and contraction force (MYH3, MYH8, ACTN3, and TNNT1) of the muscle fibers were significantly up-regulated in EOM samples of TAO, as well as those (such as VCAN, MPZ, and PTPRC) associated with cell adhesion. In addition, differentially expressed proteins related to the components and metabolism of extracellular matrix (ECM) (such as COL1A1, COL1A2, COL2A1, VCAN, OGN, and DCN) were identified. Similarly, expressions of genes involved in cell adhesion and ECM metabolism were significantly different between EOM samples of TAO patients and controls. Western blotting verified that VCAN involved in ECM proteoglycans and diseases associated with glycosaminoglycan metabolism was markedly higher in EOM samples of TAO, whereas LCN1 was obviously decreased. In conclusion, this study demonstrated the significantly altered cellular components of EOM, muscle contraction, cell adhesion and ECM metabolism, which might be involved in the pathologic mechanisms and/or consequences of TAO.

Disrupted neural signals in patients with concomitant exotropia.

PURPOSE: Decreased binocular and oculomotor function in strabismics has recently been considered as cortical in origin. This study aimed to investigate functional abnormalities using a frequency-specific neuroimaging method in patients with concomitant exotropia (XT), and to demonstrate the clinical implications. METHODS: Resting-state functional magnetic resonance imaging data were collected in 26 XT patients and 26 matched controls. To evaluate the local spontaneous neural activity, the amplitude of low frequency fluctuations (ALFF) was calculated in the typical frequency band (0.01-0.08 Hz) as well as five narrowly-defined frequency bands (slow-6: 0-0.01 Hz, slow-5: 0.01-0.027 Hz, slow-4: 0.027-0.073 Hz, slow-3: 0.073-0.167 Hz, and slow-2: 0.167-0.25 Hz), respectively. RESULTS: Patients with XT showed decreased ALFF in the bilateral parieto-occipital sulcus (POS), and increased ALFF in the bilateral thalamus within the typical frequency band. Frequency-dependent ALFF alterations were found in the higher visual areas such as the right lateral occipital complex (LOC). Furthermore, ALFF in the right LOC in the slow-5 band was positively correlated with fusion control score (r = 0.70, p < 0.0001) and binocular function score (r = 0.67, p = 0.0002). Regression analyses showed that early age of onset remained the only significant explanatory factor for ALFF reduction in the right POS in the typically-measured frequency band (also referred to as the typical frequency band) (Odds ratio, 0.038; 95% confidence interval, 0.001 to 0.075). CONCLUSIONS: Our findings provide spatial information regarding the functionally disrupted regions in XT. Moreover, the frequency-dependent ALLF alteration in the right LOC might reflect a potential plastic capacity in binocular function, which could be a potential objective index for evaluating disease severity.

The Type and Content of Collagen Fibers of the Levator Aponeurosis in Patients With Simple Congenital Blepharoptosis.

BACKGROUND: The pathogenesis of congenital blepharoptosis remains controversial and most of the studies focused on the histologic changes of the levator muscle. However, thickening of the aponeurosis was reported in congenital blepharoptosis. And the thickness of the levator aponeurosis was found to be correlated with the levator function in congenital dysmyogenic blepharoptosis. PURPOSE: The authors conducted this research to investigate the histological changes of the levator aponeurosis of simple congenital blepharoptosis patients. PATIENTS AND METHODS: The authors analyzed 12 levator aponeurosis from ptosis patients and 21 levator aponeurosis from the cadaver specimens without relative medical history. The specimens were stained with hematoxylin and eosin as well as Sirius red. The sections were examined under light-microscopy. The content and type of collagen fibers were examined with the photos taken of the sections stained with Sirius red under polarized light-microscopy. RESULTS: The content and proportion of type I collagen increased significantly in the patient group compared with the cadaver group. The proportion of type I collagen was 97.5% in the patient group, while it was 51.5% in the cadaver group. However, no correlation was found either between the levator strength, degree of ptosis and the proportion of type I collagen in the patient group or between age, gender and proportion of type I collagen in the cadaver group. CONCLUSIONS: To our knowledge, this is the first time this phenomenon was discovered. The increase of type I collagen in the levator aponeurosis of the simple congenital blepharoptosis patients may imply the increased stiffness and undermined ability of transmitting forces, dissipate energy and prevent mechanical failure in the levator aponeurosis.

Intermediate filaments in the medial rectus muscles in patients with concomitant exotropia.

PURPOSE: Distribution of intermediate filament (IF) proteins in normal extraocular muscles (EOMs) showed that the EOMs differ significantly from the other muscles in the body with respect to their IFs composition, including desmin and nestin. The aim of the present study was to investigate the pathological changes in the medial rectus (MR) in patients with concomitant exotropia (XT). METHODS: Forty-six MR muscle samples from 46 patients with XT were analyzed pathologically and processed for immunohistochemistry with specific antibodies against desmin and nestin. RESULTS: Although most of MR muscles remained normal structures relatively, they presented high expression of desmin, and in contrast, nestin was absent in a large proportion of the MR muscles. CONCLUSION: Desmin, which is downregulated in normal EOMs, had high expression in MR muscles of patients with XT. Nestin, which is present in a high proportion of normal EOMs, was downregulated in MR muscles of patients with XT.

Gene expression profile of extraocular muscles following resection strabismus surgery.

This paper aims to identify key biological processes triggered by resection surgery in the extraocular muscles (EOMs) of a rabbit model of strabismus surgery by studying changes in gene expression. Resection surgery was performed in the superior rectus of 16 rabbits and a group of non-operated rabbits served as control. Muscle samples were collected from groups of four animals 1, 2, 4 and 6 weeks after surgery and processed for RNA-sequencing and immunohistochemistry. We identified a total of 164; 136; 64 and 12 differentially expressed genes 1, 2, 4 and 6 weeks after surgery. Gene Ontology enrichment analysis revealed that differentially expressed genes were involved in biological pathways related to metabolism, response to stimulus mainly related with regulation of immune response, cell cycle and extracellular matrix. A complementary pathway analysis and network analysis performed with Ingenuity Pathway Analysis tool corroborated and completed these findings. Collagen I, fibronectin and versican, evaluated by immunofluorescence, showed that changes at the gene expression level resulted in variation at the protein level. Tenascin-C staining in resected muscles demonstrated the formation of new tendon and myotendinous junctions. These data provide new insights about the biological response of the EOMs to resection surgery and may form the basis for future strategies to improve the outcome of strabismus surgery.

Distinguishing IgG4-Related Ophthalmic Disease From Graves Orbitopathy.

PURPOSE: The authors aimed to determine key features of IgG4-related ophthalmic disease (IgG4-ROD) and Graves orbitopathy (GO) to aid in diagnosis. METHODS: The authors retrospectively identified ophthalmology patients seen between June 2009 and November 2013 with clinical overlap of GO and IgG4-ROD. Patient findings were reviewed to characterize the 2 conditions. RESULTS: Among 8 patients (7 male and 1 female), the mean age was 45.8 years. Time between diagnoses of GO and IgG4-ROD ranged from 1 month to 8 years. Imaging showed enlarged extraocular muscles in all patients. Enlarged infraorbital nerves were seen in 4 patients. Tissue biopsy showed CD20+ lymphocytes with a large proportion of IgG4 plasma cells in 7 of 8 orbital specimens. Six patients had a ratio of IgG4:IgG cells >40%. DISCUSSION: No pathognomonic clinical findings for GO or IgG4-ROD have been reported, but some key features can help distinguish the conditions. GO is likely if findings include increased thyrotropin receptor antibodies, lid retraction/lid lag, and enlarged extraocular muscles with typical tendon-sparing morphology. Findings suggestive of IgG4-ROD include history of asthma and progressive orbital disease in patients with previous diagnosis of GO, disproportionately large lateral rectus muscle, and enlarged infraorbital nerves. Increased serum IgG4 level and biopsy showing >10 IgG4+ plasma cells/high-power field and IgG4:IgG ratio >40% will support the diagnosis of IgG4-ROD. CONCLUSIONS: GO and IgG4-ROD are complicated inflammatory processes affecting the orbit and present diagnostic challenges. The authors recommend biopsy for patients who do not follow the usual clinical course of GO or have clinical characteristics of IgG4-ROD.

Histological changes underlying bupivacaine's effect on extra ocular muscle.

To determine the changes in the cross-sectional area (CSA) of myofibers and their subtype distribution based on the myosin isoform expression after bupivacaine (BUP) injection in the EOM of rabbits and help the understanding of strabismus correction after BUP injection in the clinical practice. A total of 32 rabbits received 0.3 mL of 1.5% BUP in the superior rectus muscle (SR) of the right eye (OD) and were sacrificed at days 7, 28, 60, and 92. Additional eight untouched rabbits were included as controls. Hematoxylin and eosin staining was performed, and ImageJ software was used to measure CSA. Immunohistochemical analysis was performed to analyze the proportion of myofibers positive for myosin types 1 (slow), 2 (fast) and embryonic. Myofiber area measurement decreased 7 days after BUP injection [SR, 1271 ±â€¯412 µm2 (control) to 909 ±â€¯255 µm2 (day 7)] after BUP injection, followed by an increasing trend after 28 days and normalization after 92 days [SR; 1062 ±â€¯363 µm2 (day 28), 1492 ±â€¯404 µm2 (day 60), 1317 ±â€¯334 µm2 (day 92)]. The proportion of slow myosin-positive fibers increased in the 60-day group (88.5% ±â€¯16.2%). There was no statistically significant difference in fast myosin-positive fibers. The inferior rectus of both eyes showed an increase in CSA. No increase of endomysial fibrous tissue was observed after 60 and 92 days of BUP injection. Bupivacaine, when injected into the SR of rabbits, initially decreases the fiber area followed by a transient increasing trend and normalization. There is a transient increase in the proportion of slow myosin-positive fibers in the injected muscle. Muscle adaptation in untreated EOM was found with increased CSA. These findings help clarify the clinical effects of BUP in extraocular muscle.

Effects of retinoic acid signaling on extraocular muscle myogenic precursor cells in vitro.

One major difference between limb and extraocular muscles (EOM) is the presence of an enriched population of Pitx2-positive myogenic precursor cells in EOM compared to limb muscle. We hypothesize that retinoic acid regulates Pitx2 expression in EOM myogenic precursor cells and that its effects would differ in leg muscle. The two muscle groups expressed differential retinoic acid receptor (RAR) and retinoid X receptor (RXR) levels. RXR co-localized with the Pitx2-positive cells but not with those expressing Pax7. EOM-derived and LEG-derived EECD34 cells were treated with vehicle, retinoic acid, the RXR agonist bexarotene, the RAR inverse agonist BMS493, or the RXR antagonist UVI 3003. In vitro, fewer EOM-derived EECD34 cells expressed desmin and fused, while more LEG-derived cells expressed desmin and fused when treated with retinoic acid compared to vehicle. Both EOM and LEG-derived EECD34 cells exposed to retinoic acid showed a higher percentage of cells expressing Pitx2 compared to vehicle, supporting the hypothesis that retinoic acid plays a role in maintaining Pitx2 expression. We hypothesize that retinoic acid signaling aids in the maintenance of large numbers of undifferentiated myogenic precursor cells in the EOM, which would be required to maintain EOM normalcy throughout a lifetime of myonuclear turnover.

Expression of MyoD, insulin like growth factor binding protein, thioredoxin and p27 in secondarily overacting inferior oblique muscles with superior oblique palsy.

BACKGOUND: To identify and compare specific protein levels between overacting inferior oblique (IO) muscles in superior oblique (SO) palsy patients and normal IO muscles. METHODS: We obtained 20 IO muscle samples from SO palsy patients with IO overaction ≥ + 3 who underwent IO myectomies (IOOA group), and 20 IO samples from brain death donors whose IO had functioned normally, according to their ophthalmological chart review (control group). We used MyoD for identifying satellite cell activation, insulin-like growth factor binding protein 5 (IGFBP5) for IGF effects, thioredoxin for oxidative stress, and p27 for satellite cell activation or oxidative stress in both groups. Using immunohistochemistry and Western blot, we compared expression levels of the four proteins (MyoD, IGFBP5, thioredoxin, and p27). RESULTS: Levels of thioredoxin and p27 were decreased significantly in the IOOA group. MyoD and IGFBP5 levels showed no significant difference between the groups. CONCLUSIONS: Based on these findings, the overacting IOs of patients with SO palsy had been under oxidative stress status versus normal IOs. Pathologically overacting extraocular muscles may have an increased risk of oxidative stress compared with normal extraocular muscles.

[The influence of IGF-â on the expression of Myf5 and TGF-ß1 in medial rectus muscle of cat model with strabismus].

Objective: To observe the influence of exogenous insulin-like growth factor-Ⅰ(IGF-Ⅰ) on the expression of myocyte differentiation factor 5 (Myf5) and transforming growth factor ß1(TGF-ß1) in medial rectus muscle of cat model with strabismus. Methods: Experiment research. Twenty-seven kittens which were in sensitive period of visual development (4-6 weeks old), were randomly divided into experimental, control and blank control groups by random numbers table method. Each experimental group was further divided into 3 sub-groups (4 weeks, 8 weeks and 12 weeks) based on drug intervention time, hence 3 kittens in each sub-group. The control group and the blank control group were also divided into 3 sub-groups respectively. Exotropia treatment models were set up through surgical methods and injection of IGF-Ⅰ(0.05 ml,0.1 g/L). The internal rectus muscles of 4 weeks sub-group, 8 weeks sub-group and 12 weeks sub-group were taken respectively after the treatment model had been set up. The internal rectus muscles of the control group and the blank control group were also taken according to corresponding time. The expressions of Myf5 and TGF-ß1 were tested with immunohistochemistry staining method, and optical density analysis method were employed to measure the average optical density value. The expression of Myf5 and TGF-ß1 was analyzed by Kruskal-Wallis and Bonferroni test. The correlation between the expression of Myf5 and TGF-ß1 and the time of drug intervention was analyzed by simple linear regression. Results: (1) In the experimental group, the expression of the Myf5 of the 4 weeks sub-group, 8 weeks sub-group and 12 weeks sub-group were 33.34±17.16, 39.24±15.25 and 47.70±19.39, which were higher than the control group (21.30±7.44, 19.43±4.75, 4.82±2.66) and the blank control group (18.95±6.59, 18.00±7.29, 5.86±2.61) at the same time point, and the differences were statistically significant in 8 weeks sub-group and 12 weeks sub-group (χ(2)=21.864, 31.814, both P<0.01). The expression of Myf5 in the experimental group increased with the extension of IGF-Ⅰ intervention time (R(2)=0.99, P<0.05). But there were negative correlation between expression of Myf5 and drug intervention times in the control group and the blank control group (R(2)=0.81, 0.80, both P<0.05). (2) In the experimental group, the expression of the TGF-ß1 of the 4 weeks sub-group, 8 weeks sub-group and 12 sub-weeks group were 0.80±0.12, 0.53±0.09, 0.42±0.08, which were higher than the control group (1.91±0.23, 2.30±1.03, 1.82±0.72) and the blank control group (2.01±0.31, 2.62±1.11, 1.83±0.67) at the same time point, and the differences were statistically significant (χ(2)=30.801, 40.278, 35.177, all P<0.01). The expression of TGF-ß1 in the experimental group decreased with the extension of IGF-Ⅰintervention time (R(2)=0.83, P<0.05). The average optical density value regression equation of TGF-ß1 in the sterile water control group and the blank control group was 0.04 and 0.06, respectively, and the fitting degree was very poor. Therefore, there was no correlation trend with time. Conclusions: Exogenic IGF-Ⅰ could enhance the expression of Myf5 in medial rectus muscle of cat model with strabismus. Exogenic IGF-Ⅰ could inhibit the expression of TGF-ß1 in medial rectus muscle of cat model with strabismus. Repeated injection of exogenous IGF-Ⅰ may continuously enhance the expression of Myf5 and inhibit the expression of TGF-ß1. (Chin J Ophthalmol, 2018, 54: 375-382).