Extraocular muscle satellite cells are high performance myo-engines retaining efficient regenerative capacity in dystrophin deficiency.
ABSTRACT: Extraocular muscles (EOMs) are highly specialized skeletal muscles that originate from the head mesoderm and control eye movements. EOMs are uniquely spared in Duchenne muscular dystrophy and animal models of dystrophin deficiency. Specific traits of myogenic progenitors may be determinants of this preferential sparing, but very little is known about the myogenic cells in this muscle group. While satellite cells (SCs) have long been recognized as the main source of myogenic cells in adult muscle, most of the knowledge about these cells comes from the prototypic limb muscles. In this study, we show that EOMs, regardless of their distinctive Pax3-negative lineage origin, harbor SCs that share a common signature (Pax7(+), Ki67(-), Nestin-GFP(+), Myf5(nLacZ+), MyoD-positive lineage origin) with their limb and diaphragm somite-derived counterparts, but are remarkably endowed with a high proliferative potential as revealed in cell culture assays. Specifically, we demonstrate that in adult as well as in aging mice, EOM SCs possess a superior expansion capacity, contributing significantly more proliferating, differentiating and renewal progeny than their limb and diaphragm counterparts. These robust growth and renewal properties are maintained by EOM SCs isolated from dystrophin-null (mdx) mice, while SCs from muscles affected by dystrophin deficiency (i.e., limb and diaphragm) expand poorly in vitro. EOM SCs also retain higher performance in cell transplantation assays in which donor cells were engrafted into host mdx limb muscle. Collectively, our study provides a comprehensive picture of EOM myogenic progenitors, showing that while these cells share common hallmarks with the prototypic SCs in somite-derived muscles, they distinctively feature robust growth and renewal capacities that warrant the title of high performance myo-engines and promote consideration of their properties for developing new approaches in cell-based therapy to combat skeletal muscle wasting.
Project description:Extraocular muscles (EOM) represent a unique muscle group that controls eye movements and originates from head mesoderm, while the more typically studied body and limb muscles are somite-derived. Aiming to investigate myogenic progenitors (satellite cells) in EOM versus limb and diaphragm of adult mice, we have been using flow cytometry in combination with myogenic-specific Cre-loxP lineage marking for cell isolation. While analyzing cells from the EOM of mice that harbor Myf5(Cre)-driven GFP expression, we identified in addition to the expected GFP(+) myogenic cells (presumably satellite cells), a second dominant GFP(+) population distinguished as being Sca1(+), non-myogenic, and exhibiting a fibro/adipogenic potential. This unexpected population was not only unique to EOM compared to the other muscles but also specific to the Myf5(Cre)-driven reporter when compared to the MyoD(Cre) driver. Histological studies of periocular tissue preparations demonstrated the presence of Myf5(Cre)-driven GFP(+) cells in connective tissue locations adjacent to the muscle masses, including cells in the vasculature wall. These vasculature-associated GFP(+) cells were further identified as mural cells based on the presence of the specific XLacZ4 transgene. Unlike the EOM satellite cells that originate from a Pax3-negative lineage, these non-myogenic Myf5(Cre)-driven GFP(+) cells appear to be related to cells of a Pax3-expressing origin, presumably derived from the neural crest. In all, our lineage tracing based on multiple reporter lines has demonstrated that regardless of common ancestral expression of Myf5, there is a clear distinction between periocular myogenic and non-myogenic cell lineages according to their mutually exclusive antecedence of MyoD and Pax3 gene activity.
Project description:The extraocular muscles (EOMs) are a distinct muscle group that displays an array of unique contractile, structural, and regenerative properties. They also have differential sensitivity to certain diseases and are enigmatically spared in Duchenne muscular dystrophy (DMD). The EOMs are so distinct from other skeletal muscles that the term "allotype" has been coined to highlight EOM group-specific properties. We hypothesized that increased and distinct stem cells may underlie the continual myogenesis noted in EOM. The side population (SP) stem cells were isolated and studied. EOMs had 15x higher SP cell content compared with limb muscles. Expression profiling revealed 348 transcripts that define the EOM-SP transcriptome. Over 92% of transcripts were SP specific, because they were absent in previous whole muscle microarray studies. Cultured EOM-SP cells revealed superior in vitro proliferative capacity. Finally, assays of the committed progenitors or satellite cells performed on myofibers isolated from EOM and limb muscles independently validated the increased proliferative capacity of these muscles. We suggest a model in which unique EOM stem cells contribute to the continual myogenesis noted in EOM and consistent with a role for their sparing in DMD. We believe the greater numbers of stem cells, their unique transcriptome, the greater proliferative capacity of EOM stem cells, and the greater number of satellite cells also offer clues for novel cell-based therapeutic strategies.
Project description:The pathophysiology of amyotrophic lateral sclerosis (ALS) is very complex and still rather elusive but in recent years evidence of early involvement of the neuromuscular junctions (NMJs) has accumulated. We have recently reported that the human extraocular muscles (EOMs) are far less affected than limb muscles at the end-stage of ALS from the same donor. The present study aimed to compare the differences in synaptic protein composition at NMJ and in nerve fibers between EOM and limb muscles from ALS donors and controls. Neurofilament light subunit and synaptophysin decreased significantly at NMJs and in nerve fibers in limb muscles with ALS whereas they were maintained in ALS EOMs. S100B was significantly decreased at NMJs and in nerve fibers in both EOMs and limb muscles of ALS donors, but other markers confirmed the presence of terminal Schwann cells in these NMJs. p75 neurotrophin receptor was present in nerve fibers but absent at NMJs in ALS limb muscles. The EOMs were able to maintain the integrity of their NMJs to a very large extent until the end-stage of ALS, in contrast to the limb muscles. Changes in Ca(2+) homeostasis, reflected by altered S100B distribution, might be involved in the breakdown of nerve-muscle contact at NMJs in ALS.
Project description:The skeletal fibers have different embryological origin; the extraocular and jaw-closer muscles develop from prechordal mesoderm while the limb and trunk muscles from somites. These different origins characterize also the adult muscle stem cells, known as satellite cells (SCs) and responsible for the fiber growth and regeneration. The physiological properties of presomitic SCs and their epigenetics are poorly studied despite their peculiar characteristics to preserve muscle integrity during chronic muscle degeneration. Here, we isolated SCs from canine somitic [somite-derived muscle (SDM): vastus lateralis, rectus abdominis, gluteus superficialis, biceps femoris, psoas] and presomitic [pre-somite-derived muscle (PSDM): lateral rectus, temporalis, and retractor bulbi] muscles as myogenic progenitor cells from young and old animals. In addition, SDM and PSDM-SCs were obtained also from golden retrievers affected by muscular dystrophy (GRMD). We characterized the lifespan, the myogenic potential and functions, and oxidative stress of both somitic and presomitic SCs with the aim to reveal differences with aging and between healthy and dystrophic animals. The different proliferation rate was consistent with higher telomerase activity in PSDM-SCs compared to SDM-SCs, although restricted at early passages. SDM-SCs express early (Pax7, MyoD) and late (myosin heavy chain, myogenin) myogenic markers differently from PSDM-SCs resulting in a more efficient and faster cell differentiation. Taken together, our results showed that PSDM-SCs elicit a stronger stem cell phenotype compared to SDM ones. Finally, myomiR expression profile reveals a unique epigenetic signature in GRMD SCs and miR-206, highly expressed in dystrophic SCs, seems to play a critical role in muscle degeneration. Thus, miR-206 could represent a potential target for novel therapeutic approaches.
Project description:Binocular vision requires intricate control of eye movement to align overlapping visual fields for fusion in the visual cortex, and each eye is controlled by 6 extraocular muscles (EOMs). Disorders of EOMs are an important cause of symptomatic vision loss. Importantly, EOMs represent specialized skeletal muscles with distinct gene expression profile and susceptibility to neuromuscular disorders. We aim to investigate and describe the anatomy of adult zebrafish extraocular muscles (EOMs) to enable comparison with human EOM anatomy and facilitate the use of zebrafish as a model for EOM research. Using differential interference contrast (DIC), epifluorescence microscopy, and precise sectioning techniques, we evaluate the anatomy of zebrafish EOM origin, muscle course, and insertion on the eye. Immunofluorescence is used to identify components of tendons, basement membrane and neuromuscular junctions (NMJs), and to analyze myofiber characteristics. We find that adult zebrafish EOM insertions on the globe parallel the organization of human EOMs, including the close proximity of specific EOM insertions to one another. However, analysis of EOM origins reveals important differences between human and zebrafish, such as the common rostral origin of both oblique muscles and the caudal origin of the lateral rectus muscles. Thrombospondin 4 marks the EOM tendons in regions that are highly innervated, and laminin marks the basement membrane, enabling evaluation of myofiber size and distribution. The NMJs appear to include both en plaque and en grappe synapses, while NMJ density is much higher in EOMs than in somatic muscles. In conclusion, zebrafish and human EOM anatomy are generally homologous, supporting the use of zebrafish for studying EOM biology. However, anatomic differences exist, revealing divergent evolutionary pressures.
Project description:The fast and constant activity of the extraocular muscles (EOMs) impose mechanical and metabolic stresses not typically seen in limb skeletal muscles. These functional properties may explain why EOMs seem to age at a faster rate than other skeletal muscles. Using high-density cDNA microarrays, this study investigated the gene expression profile of EOMs and extensor digitorum longus muscles (EDL) of Fischer 344/Brown Norway F1 hybrid rats at 6-, 18- and 30-months of age. At 6-mo, 705 genes and expressed sequence tags (ESTs) were differentially expressed in EOMs (436 up, 269 down). Overall, the EOM profile at this age was mostly consistent with the increased expression of fetal, developmental and EOM-specific myosin isoforms, enzymes involved in glycolysis and TCA cycle, and ion transporters and pumps, confirming the notion that EOM may represent a distinct muscle group (PNAS 98:12062, 2001). Interestingly, at 18-mo only 36 probes were significantly different in EOM (15 up, 21 down), most of them ESTs. However, at 30-mo EOMs had 655 differentially expressed genes and ESTs (480 up, 175 down). In this age group, the EOM expression profile reverted to a pattern similar to that found at 6-mo, with evidence of ongoing tissue remodeling and increased expression of antioxidant enzymes. These results indicate that the gene expression profile of EOM and EDL evolve differently throughout the lifespan. Experiment Overall Design: Total RNA was obtained with TRIzol (Invitrogen Carlsbad, CA) following the manufacturers recommended protocol. Tissues from 4 animals were combined into each RNA sample to decrease inter-subject variability. Biotinylated cRNA samples were hybridized to Affymetrix Rat Genome U34 gene chips (n=24 chips) described previously [McMullen et al. 2004]. Microarrays were washed and stained with a streptavidin-bound marker and scanned. Data were analyzed with Affymetrix Microarray Suite 5.0 software. Only genes with consistent absent/present calls in all four independent replicates per group were considered for further analysis. Comparisons the one-sided Wilcoxonâs signed rank test to estimate âincrease/no change/ decreaseâ difference calls for each pair-wise comparison. Only difference calls consistent in all pair-wise comparisons and with average changes > 2.00 were considered significant, resulting in a conservative list of genes with changed expression levels. Functional classification of genes was based on an extensive literature review.
Project description:Extraocular muscles (EOMs) show resistance to muscle dystrophies and sarcopenia. It has been recently demonstrated that they are endowed with different types of myogenic cells, all of which present an outstanding regenerative potential. Neurotrophins are important modulators of myogenic regeneration and act promoting myoblast proliferation, enhancing myogenic fusion rates and protecting myotubes from inflammatory stimuli. Here, we adapted the pre-plate cell isolation technique to obtain myogenic progenitors from the rat EOMs, and quantified their in vitro expression of neurotrophins and their receptors by RT-qPCR and immunohistochemistry, respectively. The results were compared with the expression on progenitors isolated from buccinator, tongue and limb muscles. Our quantitative analysis of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) transcripts showed, for the first time, that EOMs-derived cells express more of these factors and that they expressed TrkA, but not TrkB and TrkC receptors. On the contrary, the immunofluorescence analysis demonstrated high expression of p75NTR on all myogenic progenitors, with the EOMs-derived cells showing higher expression. Taken together, these results suggest that the intrinsic trophic differences between EOMs-derived myogenic progenitors and their counterparts from other muscles could explain why those cells show higher proliferative and fusion rates, as well as better regenerative properties.
Project description:<h4>Purpose</h4>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.<h4>Methods</h4>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.<h4>Results</h4>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.<h4>Conclusions</h4>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.
Project description:The extraocular muscles (EOMs) are a unique group of muscles that are anatomically and physiologically distinct from other muscles. We and others have shown that EOMs have a unique transcriptome and proteome. Here we investigated the expression pattern of microRNAs (miRNAs), as they may play a role in generating the unique EOM allotype. We isolated RNA and screened LC Sciences miRNA microarrays covering the sequences of miRBase 10.0 to define the microRNAome of normal mouse EOM and tibialis anterior (TA) limb muscle. Seventy-four miRNAs were found to be differentially regulated (P value <0.05) of which 31 (14 upregulated, 17 downregulated) were differentially regulated at signal strength >500. Muscle-specific miRNAs miR-206 and miR-499 were upregulated and miR-1, miR-133a, and miR-133b were downregulated in EOM. Quantitative PCR (qPCR) analysis was used to validate the differential expression. Bioinformatic tools were used to identify potential miRNA-mRNA-protein interactions and integrate data with previous transcriptome and proteomic profiling data. Luciferase assays using cotransfection of precursor miRNAs with reporter constructs containing the 3'-untranslated region of predicted target genes were used to validate targeting by identified miRNAs. The definition of the EOM microRNAome complements existing transcriptome and proteome data about the molecular makeup of EOM and provides further insight into regulation of muscle genes. These data will also help to further explain the unique EOM muscle allotype and its differential sensitivity to diseases such as Duchenne muscular dystrophy and may assist in development of therapeutic strategies.
Project description:The orbicularis oculi are the sphincter muscles of the eyelids and are involved in modulating facial expression. They differ from both limb and extraocular muscles (EOMs) in their histology and biochemistry. Weakness of the orbicularis oculi muscles is a feature of neuromuscular disorders affecting the neuromuscular junction, and weakness of facial muscles and ptosis have also been described in patients with mutations in the ryanodine receptor gene. Here, we investigate human orbicularis oculi muscles and find that they are functionally more similar to quadriceps than to EOMs in terms of excitation-contraction coupling components. In particular, they do not express the cardiac isoform of the dihydropyridine receptor, which we find to be highly expressed in EOMs where it is likely responsible for the large depolarization-induced calcium influx. We further show that human orbicularis oculi and EOMs express high levels of utrophin and low levels of dystrophin, whereas quadriceps express dystrophin and low levels of utrophin. The results of this study highlight the notion that myotubes obtained by explanting satellite cells from different muscles are not functionally identical and retain the physiological characteristics of their muscle of origin. Furthermore, our results indicate that sparing of facial and EOMs in patients with Duchenne muscular dystrophy is the result of the higher levels of utrophin expression.