Muscle Fibers Lacking Desmin in the Extraocular Muscles: A Paradigm Shift

The extraocular muscles are highly specialized muscles responsible for the complex movements of the eyeball. They differ from other skeletal muscles in many respects, including fundamental components of the contractile apparatus and the extracellular matrix. Using immunohistochemistry and a battery of well-characterized antibodies, we have investigated the composition of the cytoskeleton of their myofibers with respect to desmin, vimentin, and nestin. In the adult and fetal human extraocular muscles, a subgroup of the slow tonic muscle fibers is lacking desmin. These fibers, which are multiply innervated, show a normal myofibrillar arrangement, maintained mitochondrial distribution, and sarcolemma integrity. Desmin, the most abundant intermediate filament protein in muscle, has been considered a ubiquitous protein in skeletal muscle fibers where it links adjacent myofibrils and the myofibrillar network to the sarcolemma, the mitochondria and the membrane of the nuclei. The functional implications of the lack of desmin remain to be determined, but these findings represent a paradigm shift, as desmin has been regarded a ubiquitous protein of the cytoskeleton of muscle fibers

The Extraocular Muscles Are Selectively Spared in ALS

The extraocular muscles differ from other skeletal muscles in many respects but most strikingly in their response to neuromuscular diseases expected to affect the whole body. Oculomotor disturbances are not typical features of ALS. Recent data ascribe the muscle tissue an important role in the pathophysiology of ALS, with early involvement of the neuromuscular junctions and loss of axonal contact. We show that the extraocular muscles of terminal ALS donors and also of mice models of ALS maintain their morphology and well-preserved neuromuscular junctions until the end stages of the disease, whereas the limb muscles are severely affected and their neuromuscular junctions start losing contact with the supplying axons early in the course of ALS. There are intrinsic differences between the extraocular and limb muscles with respect to neurotrophic factors and Wnt isoforms and fundamental differences in their response to ALS that cannot be explained by the aging process. We propose that these differences may be instrumental in the selective sparing of the extraocular muscles in ALS

Protein differences between human trapezius and vastus lateralis muscles determined with a proteomic approach

Background: The trapezius muscle is a neck muscle that is susceptible to chronic pain conditions associated with repetitive tasks, commonly referred to as chronic work-related myalgia, hence making the trapezius a muscle of clinical interest. To provide a basis for further investigations of the proteomic traits of the trapezius muscle in disease, two-dimensional difference gel electrophoresis (2D-DIGE) was performed on the healthy trapezius using vastus lateralis as a reference. To obtain as much information as possible from the vast proteomic data set, both one-way ANOVA, with and without false discovery rate (FDR) correlation, and partial least square projection to latent structures with discriminant analysis (PLS-DA) were combined to compare the outcome of the analysis. Results: The trapezius and vastus lateralis showed significant differences in metabolic, contractile and regulatory proteins, with different results depending on choice of statistical approach and pre-processing technique. Using the standard method, FDR correlated one-way ANOVA, 42 protein spots differed significantly in abundance between the two muscles. Complementary analysis using immunohistochemistry and western blot confirmed the results from the 2D-DIGE analysis. Conclusions: The proteomic approach used in the present study combining 2D-DIGE and multivariate modelling provided a more comprehensive comparison of the protein profiles of the human trapezius and vastus lateralis muscle, than previously possible to obtain with immunohistochemistry or SDS-PAGE alone. Although 2D-DIGE has inherent limitations it is particularly useful to comprehensively screen for important structural and metabolic proteins, and appears to be a promising tool for future studies of patients suffering from chronic work related myalgia or other muscle diseases

Chromatic Pupillometry in Children

Chromatic pupillometry is a technique that is increasingly used to assess retinal disorders. As age may be one of the various factors which can influence the pupillary light reaction, this study aimed to evaluate the pupil responses to colored light stimuli in the pediatric population. Fifty-three children with normal vision and without any history of ocular disorders were tested with a portable pupillometer. Four test sequences were used: five dim blue (470 nm) stimuli presented in half log steps ranging from −3.15 to −1.15 log cd/m2 after 3 min of dark adaptation, five red (622 nm) stimuli of −1.15, −0.7, −0.15, 0.3, and 0.85 log cd/m2 after 1 min light adaptation, one bright blue stimulus of 2.2 log cd/m2 and one bright red of 2 log cd/m2. The results were grouped by age: a younger group included 27 children aged from 3 to 10 years old and an older group included 26 from 10 and 1 month to 18 years old. The younger group had a smaller pupil diameter after dark adaptation compared with the older group. A linear regression defining the photopic threshold showed that younger subjects had a higher threshold, e.g., needed a brighter red stimulus to evoke a threshold pupil response comparable that of subjects. Age thus seems to influence outer retinal sensitivity at least as evaluated by the pupillary photopic threshold intensity. The post-illumination pupillary reaction was used as a marker of intrinsic melanopsin activity and did not show any difference between the two age groups

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

A novel type of multiterminal motor endplate in human extraocular muscles

Purpose: To investigate the relation between type of motor endplate, acetylcholine receptor (AChR) subunit composition, and fiber types in human extraocular muscles (EOMs). Methods: EOM samples collected from subjects aged 34 to 82 years were serially sectioned and processed for immunohistochemistry, with specific antibodies against different myosin heavy chain (MyHC) isoforms, neurofilament, synaptophysin, and adult epsilon (ε) and fetal gamma (γ) AChR subunits as well as α-bungarotoxin. Results: A novel type of motor endplate consisting of large, multiterminal en plaque endings was found in human EOMs, in addition to the previously well-described single en plaque and multiple en grappe endplates. Such novel endplates were abundant but exclusively observed in myofibers lacking MyHC slow and fast IIa but containing MyHC extraocular (MyHCeom), isoforms. Multiple en grappe endings were found only in myofibers containing MyHC slow-tonic isoform and contained fetal γ AChR subunit. Adult ε and fetal γ AChR subunits, alone or combined, were found in the multiterminal endplates. Distinct AChR subunits were present in adjacent motor endplates of a given myofiber containing MyHCeom. Conclusions: Human EOMs have a more complex innervation pattern than previously described, comprising also a novel type of multiterminal motor endplate present in myofibers containing MyHCeom. The heterogeneity in AChR subunit composition in a given myofiber suggests the possible presence of polyneuronal innervation in human EOMs

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 alpha-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

Altered Signaling Pathways in Aniridia-Related Keratopathy

PURPOSE. To study the Notch1, Wnt/beta-catenin, sonic hedgehog (SHH), and mammalian target of rapamycin (mTOR) cell signaling pathways in naive and surgically treated corneas of aniridia cases with advanced aniridia-related keratopathy (ARK). METHODS. Two naive corneal buttons from patients with advanced ARK submitted to penetrating keratoplasty for the first time, one corneal button from an ARK patient that had undergone a keratolimbal allograft (KLAL), two corneal buttons from ARK patients who had previously undergone centered or decentered transplantation, and two adult healthy control corneas were processed for immunohistochemistry in this descriptive study. Antibodies specific against elements of the Notch1 (Notch1; Dlk1; Numb), Wnt/beta-catenin (Wnt5a; Wnt7a; beta-catenin), SHH (glioma-associated oncogene homolog [Gli1]; Hes1), and mTOR (mTOR1; ribosomal protein S6 [rpS6]) signaling pathways were used as well as antibodies against PAX6 and keratin 13 (Krt13). RESULTS. All ARK corneas presented signs of conjunctivalization and analogous signaling pathway changes in the subepithelial pannus and epithelium, with decreased detection of the Notch1 signaling pathway and an increased presence of the Notch1 inhibitors Numb and Dlk1. Increased detections of Wnt/beta-catenin (enhanced presence of Wnt5a, Wnt7a, and beta-catenin), SHH (detection of Gli1 and Hes1), and mTOR (identification of mTOR and rpS6) signaling pathways were found in the subepithelial pannus and epithelium of all ARK corneas, when compared with normal controls. CONCLUSIONS. The similarity in pathway alterations found in all ARK corneas, irrespective of limbal stem cell transplantation, further supports the discussion on the role of host-specific factors and limbal stem cell deficiency in ARK

Understanding the extraocular muscles : Connective tissue, motor endplates and the cytoskeleton

We constantly direct our eyes to the object of interest with the help of the extraocular muscles, andthereby use foveal fixation to attain the best possible visual acuity. The muscles around the eye arerather different from other skeletal muscles, being, for example, simultaneously the fastest musclesin the body and impossible to exhaust. The most exciting property of the extraocular muscles is theirunique response to disease, as they often remain unaffected in muscle conditions which lead tosevere handicap and premature death. Understanding the coping strategies that allow the extraocularmuscles to remain unaffected may provide clues for the future treatment of severe diseases such asmuscle dystrophies

Chromatic pupillometry in children

Chromatic pupillometry is a technique that is increasingly used to assess retinal disorders. As age may be one of the various factors which can influence the pupillary light reaction, this study aimed to evaluate the pupil responses to colored light stimuli in the pediatric population. Fifty-three children with normal vision and without any history of ocular disorders were tested with a portable pupillometer. Four test sequences were used: five dim blue (470 nm) stimuli presented in half log steps ranging from -3.15 to -1.15 log cd/m(2) after 3 min of dark adaptation, five red (622 nm) stimuli of -1.15, -0.7, -0.15, 0.3, and 0.85 log cd/m(2) after 1 min light adaptation, one bright blue stimulus of 2.2 log cd/m(2) and one bright red of 2 log cd/m(2). The results were grouped by age: a younger group included 27 children aged from 3 to 10 years old and an older group included 26 from 10 and 1 month to 18 years old. The younger group had a smaller pupil diameter after dark adaptation compared with the older group. A linear regression defining the photopic threshold showed that younger subjects had a higher threshold, e.g., needed a brighter red stimulus to evoke a threshold pupil response comparable that of subjects. Age thus seems to influence outer retinal sensitivity at least as evaluated by the pupillary photopic threshold intensity. The post-illumination pupillary reaction was used as a marker of intrinsic melanopsin activity and did not show any difference between the two age groups