Effect of Fibroblast Growth Factor 2 on Extraocular Muscle Structure and Function.

Purpose: Mutations in the fibroblast growth factor (FGF) receptor can result in strabismus, but little is known about how FGFs affect extraocular muscle structure and function. These were assessed after short-term and long-term exposure to exogenously applied FGF2 to determine the effect of enhanced signaling. Methods: One superior rectus muscle of adult rabbits received either a series of three injections of 500 ng, 1 µg, or 5 µg FGF2 and examined after 1 week, or received sustained treatment with FGF2 and examined after 1, 2, or 3 months. Muscles were assessed for alterations in force generation, myofiber size, and satellite cell number after each treatment. Results: One week after the 5 µg FGF2 injections, treated muscles showed significantly increased force generation compared with naïve controls, which correlated with increased myofiber cross-sectional areas and Pax7-positive satellite cells. In contrast, 3 months of sustained FGF2 treatment resulted in decreased force generation, which correlated with decreased myofiber size and decreased satellite cells compared with naïve control and the untreated contralateral side. Conclusions: FGF2 had distinctly different effects when short-term and long-term treatments were compared. The decreased size and ability to generate force correlated with decreased myofiber areas seen in individuals with Apert syndrome, where there is sustained activation of FGF signaling. Knowing more about signaling pathways critical for extraocular muscle function, development, and disease will pave the way for improved treatment options for strabismus patients with FGF abnormalities in craniofacial disease, which also may be applicable to other strabismus patients.

Cytoskeletal Proteins in Myotendinous Junctions of Human Extraocular Muscles.

Purpose: The purpose of this study was to investigate the cytoskeletal composition of myotendinous junctions (MTJs) in the human extraocular muscles (EOMs). Desmin and other major cytoskeletal proteins are enriched at the MTJs of ordinary myofibers, where they are proposed to be of particular importance for force transmission and required to maintain myofiber integrity. Methods: EOM and limb muscle samples were analyzed with immunohistochemistry using antibodies against the intermediate filament proteins desmin, nestin, keratin 19, vimentin, and different myosin heavy chain (MyHC) isoforms. MTJs were identified by labeling with antibodies against laminin or tenascin. Results: In contrast to MTJs in lumbrical muscle where desmin, nestin, and keratin 19 were always present, approximately one-third of the MTJs in the EOMs lacked either desmin and/or nestin, and all MTJs lacked keratin 19. Approximately 6% of the MTJs in the EOMs lacked all of these key cytoskeletal proteins. Conclusions: The cytoskeletal protein composition of MTJs in human EOMs differed significantly from that of MTJs in limb muscles. These differences in cytoskeletal protein composition may indicate particular adaptation to meet the functional requirements of the EOMs.

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.

Twist3 is required for dedifferentiation during extraocular muscle regeneration in adult zebrafish.

Severely damaged adult zebrafish extraocular muscles (EOMs) regenerate through dedifferentiation of residual myocytes involving a muscle-to-mesenchyme transition. Members of the Twist family of basic helix-loop-helix transcription factors (TFs) are key regulators of the epithelial-mesenchymal transition (EMT) and are also involved in craniofacial development in humans and animal models. During zebrafish embryogenesis, twist family members (twist1a, twist1b, twist2, and twist3) function to regulate craniofacial skeletal development. Because of their roles as master regulators of stem cell biology, we hypothesized that twist TFs regulate adult EOM repair and regeneration. In this study, utilizing an adult zebrafish EOM regeneration model, we demonstrate that inhibiting twist3 function using translation-blocking morpholino oligonucleotides (MOs) impairs muscle regeneration by reducing myocyte dedifferentiation and proliferation in the regenerating muscle. This supports our hypothesis that twist TFs are involved in the early steps of dedifferentiation and highlights the importance of twist3 during EOM regeneration.

Lessons from Amphioxus Bauplan About Origin of Cranial Nerves of Vertebrates That Innervates Extrinsic Eye Muscles.

Amphioxus is the living chordate closest to the ancestral form of vertebrates, and in a key position to reveal essential aspects of the evolution of the brain Bauplan of vertebrates. The dorsal neural cord of this species at the larval stage is characterized by a small cerebral vesicle at its anterior end and a large posterior region. The latter is comparable in some aspects to the hindbrain and spinal cord regions of vertebrates. The rostral end of the cerebral vesicle contains a median pigment spot and associated rows of photoreceptor and other nerve cells; this complex is known as "the frontal eye." However, this is not a complete eye in the sense that it has neither eye muscles nor lens (only a primitive retina-like tissue). Cranial nerves III, IV, and VI take part in the motor control of eye muscles in all vertebrates. Using a recent model that postulates distinct molecularly characterized hypothalamo-prethalamic and mesodiencephalic domains in the early cerebral vesicle of amphioxus, we analyze here possible scenarios for the origin from the common ancestor of cephalochordates and vertebrates of the cranial nerves related with extrinsic eye muscle innervations. Anat Rec, 302:452-462, 2019. © 2018 Wiley Periodicals, Inc.

The Cytoskeleton in the Extraocular Muscles of Desmin Knockout Mice.

Purpose: To investigate the effect of absence of desmin on the extraocular muscles (EOMs) with focus on the structure and composition of the cytoskeleton. Methods: The distribution of synemin, syncoilin, plectin, nestin, and dystrophin was evaluated on cross and longitudinal sections of EOMs and limb muscles from 1-year-old desmin knockout mice (desmin-/-) by immunofluorescence. General morphology was evaluated with hematoxylin and eosin while mitochondrial content and distribution were evaluated by succinate dehydrogenase (SDH) and modified Gomori trichrome stainings. Results: The muscle fibers of the EOMs in desmin-/- mice were remarkably well preserved in contrast to those in the severely affected soleus and the slightly affected gastrocnemius muscles. There were no signs of muscular pathology in the EOMs and all cytoskeletal proteins studied showed a correct location at sarcolemma and Z-discs. However, an increase of SDH staining and mitochondrial aggregates under the sarcolemma was detected. Conclusions: The structure of the EOMs was well preserved in the absence of desmin. We suggest that desmin is not necessary for correct synemin, syncoilin, plectin, and dystrophin location on the cytoskeleton of EOMs. However, it is needed to maintain an appropriate mitochondrial distribution in both EOMs and limb muscles.

Changing muscle function with sustained glial derived neurotrophic factor treatment of rabbit extraocular muscle.

Recent microarray and RNAseq experiments provided evidence that glial derived neurotrophic factor (GDNF) levels were decreased in extraocular muscles from human strabismic subjects compared to age-matched controls. We assessed the effect of sustained GDNF treatment of the superior rectus muscles of rabbits on their physiological and morphological characteristics, and these were compared to naïve control muscles. Superior rectus muscles of rabbits were implanted with a sustained release pellet of GDNF to deliver 2µg/day, with the contralateral side receiving a placebo pellet. After one month, the muscles were assessed using in vitro physiological methods. The muscles were examined histologically for alteration in fiber size, myosin expression patterns, neuromuscular junction size, and stem cell numbers and compared to age-matched naïve control muscles. GDNF resulted in decreased force generation, which was also seen on the untreated contralateral superior rectus muscles. Muscle relaxation times were increased in the GDNF treated muscles. Myofiber mean cross-sectional areas were increased after the GDNF treatment, but there was a compensatory increase in expression of developmental, neonatal, and slow tonic myosin heavy chain isoforms. In addition, in the GDNF treated muscles there was a large increase in Pitx2-positive myogenic precursor cells. One month of GDNF resulted in significant extraocular muscle adaptation. These changes are interesting relative to the decreased levels of GDNF in the muscles from subjects with strabismus and preliminary data in infant non-human primates where sustained GDNF treatment produced a strabismus. These data support the view that GDNF has the potential for improving eye alignment in subjects with strabismus.

Extraocular muscle regeneration in zebrafish requires late signals from Insulin-like growth factors.

Insulin-like growth factors (Igfs) are key regulators of key biological processes such as embryonic development, growth, and tissue repair and regeneration. The role of Igf in myogenesis is well documented and, in zebrafish, promotes fin and heart regeneration. However, the mechanism of action of Igf in muscle repair and regeneration is not well understood. Using adult zebrafish extraocular muscle (EOM) regeneration as an experimental model, we show that Igf1 receptor blockage using either chemical inhibitors (BMS754807 and NVP-AEW541) or translation-blocking morpholino oligonucleotides (MOs) reduced EOM regeneration. Zebrafish EOMs regeneration depends on myocyte dedifferentiation, which is driven by early epigenetic reprogramming and requires autophagy activation and cell cycle reentry. Inhibition of Igf signaling had no effect on either autophagy activation or cell proliferation, indicating that Igf signaling was not involved in the early reprogramming steps of regeneration. Instead, blocking Igf signaling produced hypercellularity of regenerating EOMs and diminished myosin expression, resulting in lack of mature differentiated muscle fibers even many days after injury, indicating that Igf was involved in late re-differentiation steps. Although it is considered the main mediator of myogenic Igf actions, Akt activation decreased in regenerating EOMs, suggesting that alternative signaling pathways mediate Igf activity in muscle regeneration. In conclusion, Igf signaling is critical for re-differentiation of reprogrammed myoblasts during late steps of zebrafish EOM regeneration, suggesting a regulatory mechanism for determining regenerated muscle size and timing of differentiation, and a potential target for regenerative therapy.

Composition, Architecture, and Functional Implications of the Connective Tissue Network of the Extraocular Muscles.

Purpose: We examined the pattern and extent of connective tissue distribution in the extraocular muscles (EOMs) and determined the ability of the interconnected connective tissues to disseminate force laterally. Methods: Human EOMs were examined for collagens I, III, IV, and VI; fibronectin; laminin; and elastin using immunohistochemistry. Connective tissue distribution was examined with scanning electron microscopy. Rabbit EOMs were examined for levels of force transmission longitudinally and transversely using in vitro force assessment. Results: Collagens I, III, and VI localized to the endomysium, perimysium, and epimysium. Collagen IV, fibronectin, and laminin localized to the basal lamina surrounding all myofibers. All collagens localized similarly in the orbital and global layers throughout the muscle length. Elastin had the most irregular pattern and ran longitudinally and circumferentially throughout the length of all EOMs. Scanning electron microscopy showed these elements to be extensively interconnected, from endomysium through the perimysium to the epimysium surrounding the whole muscle. In vitro physiology demonstrated force generation in the lateral dimension, presumably through myofascial transmission, which was always proportional to the force generated in the longitudinally oriented muscles. Conclusions: A striking connective tissue matrix interconnects all the myofibers and extends, via perimysial connections, to the epimysium. These interconnections are significant and allow measurable force transmission laterally as well as longitudinally, suggesting that they may contribute to the nonlinear force summation seen in motor unit recording studies. This provides strong evidence that separate compartmental movements are unlikely as no region is independent of the rest of the 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.