Distinct transcriptomic profile of satellite cells contributes to preservation of neuromuscular junctions in extraocular muscles of ALS mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder characterized by progressive weakness of almost all skeletal muscles, whereas extraocular muscles (EOMs) are comparatively spared. While hindlimb and diaphragm muscles of end-stage SOD1G93A (G93A) mice (a familial ALS mouse model) exhibit severe denervation and depletion of Pax7+satellite cells (SCs), we found that the pool of SCs and the integrity of neuromuscular junctions (NMJs) are maintained in EOMs. In cell sorting profiles, SCs derived from hindlimb and diaphragm muscles of G93A mice exhibit denervation-related activation, whereas SCs from EOMs of G93A mice display spontaneous (non-denervation-related) activation, similar to SCs from wild-type mice. Specifically, cultured EOM SCs contain more abundant transcripts of axon guidance molecules, including Cxcl12, along with more sustainable renewability than the diaphragm and hindlimb counterparts under differentiation pressure. In neuromuscular co-culture assays, AAV-delivery of Cxcl12 to G93A-hindlimb SC-derived myotubes enhances motor neuron axon extension and innervation, recapitulating the innervation capacity of EOM SC-derived myotubes. G93A mice fed with sodium butyrate (NaBu) supplementation exhibited less NMJ loss in hindlimb and diaphragm muscles. Additionally, SCs derived from G93A hindlimb and diaphragm muscles displayed elevated expression of Cxcl12 and improved renewability following NaBu treatment in vitro. Thus, the NaBu-induced transcriptomic changes resembling the patterns of EOM SCs may contribute to the beneficial effects observed in G93A mice. More broadly, the distinct transcriptomic profile of EOM SCs may offer novel therapeutic targets to slow progressive neuromuscular functional decay in ALS and provide possible 'response biomarkers' in pre-clinical and clinical studies.

fhl2b mediates extraocular muscle protection in zebrafish models of muscular dystrophies and its ectopic expression ameliorates affected body muscles.

In muscular dystrophies, muscle fibers loose integrity and die, causing significant suffering and premature death. Strikingly, the extraocular muscles (EOMs) are spared, functioning well despite the disease progression. Although EOMs have been shown to differ from body musculature, the mechanisms underlying this inherent resistance to muscle dystrophies remain unknown. Here, we demonstrate important differences in gene expression as a response to muscle dystrophies between the EOMs and trunk muscles in zebrafish via transcriptomic profiling. We show that the LIM-protein Fhl2 is increased in response to the knockout of desmin, plectin and obscurin, cytoskeletal proteins whose knockout causes different muscle dystrophies, and contributes to disease protection of the EOMs. Moreover, we show that ectopic expression of fhl2b can partially rescue the muscle phenotype in the zebrafish Duchenne muscular dystrophy model sapje, significantly improving their survival. Therefore, Fhl2 is a protective agent and a candidate target gene for therapy of muscular dystrophies.

Obscurin Maintains Myofiber Identity in Extraocular Muscles.

Purpose: The cytoskeleton of the extraocular muscles (EOMs) is significantly different from that of other muscles. We aimed to investigate the role of obscurin, a fundamental cytoskeletal protein, in the EOMs. Methods: The distribution of obscurin in human and zebrafish EOMs was compared using immunohistochemistry. The two obscurin genes in zebrafish, obscna and obscnb, were knocked out using CRISPR/Cas9, and the EOMs were investigated using immunohistochemistry, qPCR, and in situ hybridization. The optokinetic reflex (OKR) in five-day-old larvae and adult obscna-/-;obscnb-/- and sibling control zebrafish was analyzed. Swimming distance was recorded at the same age. Results: The obscurin distribution pattern was similar in human and zebrafish EOMs. The proportion of slow and fast myofibers was reduced in obscna-/-;obscnb-/- zebrafish EOMs but not in trunk muscle, whereas the number of myofibers containing cardiac myosin myh7 was significantly increased in EOMs of obscurin double mutants. Loss of obscurin resulted in less OKRs in zebrafish larvae but not in adult zebrafish. Conclusions: Obscurin expression is conserved in normal human and zebrafish EOMs. Loss of obscurin induces a myofiber type shift in the EOMs, with upregulation of cardiac myosin heavy chain, myh7, showing an adaptation strategy in EOMs. Our model will facilitate further studies in conditions related to obscurin.

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.

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.

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.

Eye Movements But Not Vision Drive the Development of Palisade Endings.

Purpose: To test whether visual experience and/or eye movements drive the postnatal development of palisade endings in extraocular muscles. Methods: In three newborn cats, the right eye was covered until 30 days from postnatal (P) day 7 (before opening their eyes), and in three cats both eyes were covered until 45 days, also from P7. To block eye movements, another seven cats received a retrobulbar injection of botulinum neurotoxin A (BoNT-A) into the left orbit at birth and survived for 45 days (three cats) and 95 days (four cats). The distal third of the rectus muscles containing the palisade endings was used for whole-mount preparation and triple-fluorescence labeling with anti-neurofilament along with (1) anti-synaptophysin and phalloidin or (2) anti-growth associated protein 43 (GAP43) and phalloidin. Immunolabeled specimens were analyzed in the confocal laser scanning microscope. Results: After unilateral and bilateral dark rearing, palisade endings were qualitatively and quantitatively equal to those from age-matched controls. After BoNT-A induced eye immobilization for 45 or 95 days, palisade endings were absent in the superior rectus and lateral rectus muscles and only present in the inferior rectus and medial rectus muscle. These BoNT-A-treated palisade endings were rudimentary and reduced in number, and the expression of the neuronal developmental protein GAP43 was significantly reduced. Conclusions: This study demonstrates that eye immobilization, but not visual deprivation, affects palisade ending development. Palisade endings develop in the first month of life, and the present findings indicate that, during this time window, palisade endings are prone to oculomotor perturbations.

Increased angiogenic factors in the aqueous and vitreous humors after disinsertion of extraocular muscle and the effects of triamcinolone acetate injection.

The four extraocular rectus muscles in the rabbits were disinserted for induction of anterior segment ischemia (ASI) and the changes in the concentrations of prostaglandin E2 (PGE2), hypoxia-inducible factor-1 (HIF-1α), and vascular endothelial growth factor (VEGF) in the aqueous and vitreous humor were evaluated. Disinsertion of four rectus muscles in rabbits was performed in the right eyes of rabbits (ASI group). The concentrations of PGE2, HIF-1α, and VEGF in the aqueous and vitreous humor were measured at 1, 3, 6, 12, and 24 h by ELISA. The concentrations were compared with those of the fellow eyes (contralateral group) and normal healthy eyes (control group). Subconjunctival injection of triamcinolone acetonide (TA) was administered and three cytokine concentrations in the aqueous humor and vitreous humor were measured at 12 h after TA injection. A total of 48 eyes from 28 rabbits were included. The concentrations of PGE2, HIF-1α, and VEGF in the aqueous humor in the ASI and contralateral groups were significantly higher than those in the control group (p < 0.05, all). The aqueous and vitreous humor concentrations of VEGF in eyes with simultaneous TA injection were significantly lower than were those in the ASI group (p = 0.02, all). The concentration of PGE2, HIF-1α, and VEGF in the aqueous humor was increased after induction of ASI and TA injection seems to be effective in inhibiting VEGF elevation in ASI.

Extraocular Muscle Enlargement in Growth Hormone-Secreting Pituitary Adenomas.

BACKGROUND AND PURPOSE: While Graves disease is the most common cause of extraocular muscle enlargement, case reports have also associated growth hormone-secretory pituitary adenomas with this same phenomenon. We investigated the prevalence and response to treatment of extraocular muscle enlargement in patients with growth hormone-secretory pituitary adenomas. MATERIALS AND METHODS: We retrospectively reviewed extraocular muscle sizes using MR imaging in patients with growth hormone-secretory pituitary adenomas who underwent a transsphenoidal surgical resection compared with a matched control group with nonsecretory pituitary adenomas. Descriptive and comparative statistics were used to evaluate patient characteristics and extraocular muscle sizes between the 2 groups. RESULTS: We identified 16 patients who presented with growth hormone-secreting pituitary adenomas and underwent transsphenoidal surgical resection from 2010 to 2019. The average diameter of the extraocular muscle at the time of diagnosis for the group with growth hormone-secretory pituitary adenomas was larger than that in the control group (4.7 versus 3.8 mm, P < .001). Nine patients achieved insulin-like growth factor 1 level normalization at a median of 11.5 months before their most recent MR imaging evaluation. The average size of the extraocular muscles of patients who achieved a normalized insulin-like growth factor 1 was smaller compared with those that did not (difference, 0.7 mm; 95% CI, 0.3-1.2 mm; P < .001), approaching the size of extraocular muscle in the control group. CONCLUSIONS: We describe a high prevalence of extraocular muscle enlargement in patients with growth hormone-secreting pituitary adenomas. Additionally, we note that the size of extraocular muscles decreased with associated improvement in the biochemical control of acromegaly.

Development of Nystagmus With the Absence of MYOD Expression in the Extraocular Muscles.

Purpose: Myoblast determination protein 1 (MYOD) is a critical myogenic regulatory factor in muscle development, differentiation, myofiber repair, and regeneration. As the extraocular muscles significantly remodel their myofibers throughout life compared with limb skeletal muscles, we hypothesized that the absence of MYOD would result in their abnormal structure and function. To assess structural and functional changes in the extraocular muscles in MyoD-/- mice, fiber size and number and optokinetic nystagmus reflex (OKN) responses were examined. Methods: OKN was measured in MyoD-/- mice and littermate wild-type controls at 3, 6, and 12 months. The extraocular muscles were examined histologically for changes in mean myofiber cross-sectional area, total myofiber number, and nuclei immunostained for PAX7 and PITX2, markers of myogenic precursor cells. Results: The MyoD-/- mice developed nystagmus, with both jerk and pendular waveforms, in the absence and in the presence of moving visual stimulation. At 12 months, there were significant losses in mean myofiber cross-sectional area and in total number of orbital layer fibers in all rectus muscles, as well as in global layer fibers in the superior and inferior rectus muscles. Haploinsufficient mice showed abnormal OKN responses. PITX2-positive cell entry into myofibers of the MyoD-/- mice was significantly reduced. Conclusions: This study is the first demonstration of the development of nystagmus in the constitutive absence of expression of the muscle-specific transcription factor MYOD. We hypothesize that myofiber loss over time may alter anterograde and/or retrograde communication between the motor nerves and extraocular muscles that are critical for maintaining normalcy of extraocular muscle function.

Prognostic factors of restrictive myopathy in thyroid eye disease.

To investigate the prognostic factors of extraocular muscle restriction in patients with thyroid eye disease (TED), 65 patients with TED and restrictive myopathy were evaluated. Demographics, clinical activity score (CAS), smoking status, thyroid disease status, thyroid hormone status, thyroid autoantibody status, orbital computed tomography (CT) scan at initial presentation, and treatment regimens were assessed. The movements of the most severely affected extraocular muscles were categorized into five grades. The patients were divided into the improved and the not-improved group based on the improvement in the limitation of the extraocular muscle excursion (LOM) throughout the follow-up, and the groups were compared using clinical factors. The mean LOM significantly improved from 2.3 ± 1.1 to 1.7 ± 1.2 after 1 year of follow-up. The excursion of the most restricted muscle improved in 32 patients but not in 33 patients during the follow-up. The initial concentration of the thyroid-stimulating antibody (TSAb) was significantly lower in the improved (229.3 ± 114.1) than in the not-improved group (345.0 ± 178.6) (P = 0.02) Age, sex, smoking status, CAS, thyroid status, and muscle thickness on the CT scan did not significantly differ in the groups. This study showed that the initial concentration of TSAb is a factor affecting the recovery of restrictive myopathy.

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.

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.

Differences in relative capacities of oxidative phosphorylation pathways may explain sex- and tissue-specific susceptibility to vision defects due to mitochondrial dysfunction.

Mitochondrial dysfunction is a major cause and/or contributor to the development and progression of vision defects in many ophthalmologic and mitochondrial diseases. Despite their mechanistic commonality, these diseases exhibit an impressive variety in sex- and tissue-specific penetrance, incidence, and severity. Currently, there is no functional explanation for these differences. We measured the function, relative capacities, and patterns of control of various oxidative phosphorylation pathways in the retina, the eyecup, the extraocular muscles, the optic nerve, and the sciatic nerve of adult male and female rats. We show that the control of mitochondrial respiratory pathways in the visual system is sex- and tissue-specific and that this may be an important factor in determining susceptibility to mitochondrial dysfunction between these groups. The optic nerve showed a low relative capacity of the NADH pathway, depending on complex I, compared to other tissues relying mainly on mitochondria for energy production. Furthermore, NADH pathway capacity is higher in females compared to males, and this sexual dimorphism occurs only in the optic nerve. Our results propose an explanation for Leber's hereditary optic neuropathy, a mitochondrial disease more prevalent in males where the principal tissue affected is the optic nerve. To our knowledge, this is the first study to identify and provide functional explanations for differences in the occurrence and severity of visual defects between tissues and between sexes. Our results highlight the importance of considering sex- and tissue-specific mitochondrial function in elucidating pathophysiological mechanisms of visual defects.

Palisade Endings Have an Exocytotic Machinery But Lack Acetylcholine Receptors and Distinct Acetylcholinesterase Activity.

Purpose: The purpose of this work was to test whether palisade endings express structural and molecular features of exocytotic machinery, and are associated with acetylcholine receptors, and enzymes for neurotransmitter breakdown. Methods: Extraocular rectus muscles from six cats were studied. Whole-mount preparations of extraocular muscles (EOMs) were immunolabeled with markers for exocytotic proteins, including synaptosomal-associated protein of 25 kDa (SNAP25), syntaxin, synaptobrevin, synaptotagmin, and complexin. Acetylcholine receptors (AChRs) were visualized with α-bungarotoxin and with an antibody against AChRs, and acetylcholinesterase (AChE) was tagged with anti-AChE. Molecular features of multicolor labeled palisade endings were analyzed in the confocal scanning microscope, and their ultrastructural features were revealed in the transmission electron microscope. Results: All palisade endings expressed the exocytotic proteins SNAP25, syntaxin, synaptobrevin, synaptotagmin, and complexin. At the ultrastructural level, vesicles docked at the plasma membrane of terminal varicosities of palisade endings. No AChRs were associated with palisade endings as demonstrated by the absence of α-bungarotoxin and anti-AChR binding. AChE, the degradative enzyme for acetylcholine exhibited low, if any, activity in palisade endings. Axonal tracking showed that axons with multiple en grappe motor terminals were in continuity with palisade endings. Conclusions: This study demonstrates that palisade endings exhibit structural and molecular characteristics of exocytotic machinery, suggesting neurotransmitter release. However, AChRs were not associated with palisade endings, so there is no binding site for acetylcholine, and, due to low/absent AChE activity, insufficient neurotransmitter removal. Thus, the present findings indicate that palisade endings belong to an effector system that is very different from that found in other skeletal muscles.