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Skeletal muscle is a complex tissue containing tissue resident muscle stem cells (satellite cells) (MuSCs) important for postnatal muscle growth and regeneration. Quantitative analysis of the biological function of MuSCs and the molecular pathways responsible for a potential juxtavascular niche for MuSCs is currently lacking. We utilized fluorescent reporter mice and muscle tissue clearing to investigate the proximity of MuSCs to capillaries in 3 dimensions. We show that MuSCs express abundant VEGFA, which recruits endothelial cells (ECs) in vitro, whereas blocking VEGFA using both a vascular endothelial growth factor (VEGF) inhibitor and MuSC-specific VEGFA gene deletion reduces the proximity of MuSCs to capillaries. Importantly, this proximity to the blood vessels was associated with MuSC self-renewal in which the EC-derived Notch ligand Dll4 induces quiescence in MuSCs. We hypothesize that MuSCs recruit capillary ECs via VEGFA, and in return, ECs maintain MuSC quiescence though Dll4., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Purpose: Surgical recession of an extraocular muscle (EOM) posterior to its original insertion is a common form of strabismus surgery, weakening the rotational force exerted by the muscle on the globe and improving eye alignment. The purpose of this study was to assess myosin heavy chain (MyHC) isoform expression and satellite cell activity as defined by Pax7 expression in recessed EOMs of adult rabbits compared with that in muscles tenotomized but not recessed and with that in normal control muscles., Methods: The scleral insertion of the superior rectus muscle was detached and sutured either 7 mm posterior to its original insertion site (recession surgery) or at the same site (tenotomy). One day before euthanization, the rabbits received bromodeoxyuridine (BrdU) injections. After 7 and 14 days, selected EOMs from both orbits were examined for changes in fast, slow, neonatal, and developmental MyHC isoform expression, Pax7 expression, and BrdU incorporation., Results: Recession and tenotomy surgery resulted in similar changes in the surgical EOMs. These included a decreased proportion of fast MyHC myofibers, an increased proportion of slow MyHC myofibers, and increased BrdU-positive satellite cells. Similar changes were seen in the non-operated contralateral superior rectus muscles. The ipsilateral inferior rectus showed reciprocal changes to the surgical superior rectus muscles., Conclusions: The EOMs are extremely adaptive to changes induced by recession and tenotomy surgery, responding with modulations in fiber remodeling and myosin expression. These adaptive responses could be manipulated to improve surgical success rates.

Myoblast transfer therapy for Duchenne muscular dystrophy (DMD) largely fails due to cell death and inability of transplanted cells to engraft in diseased muscles. One method attempting to enrich for cell subpopulations is the Hoechst 33342 dye exclusion assay, yielding a side population (SP) thought to be progenitor enriched and a main population (MP). However, in vitro and transplant studies yielded inconsistent results relative to downstream progeny. Cell surface markers expressed by skeletal muscle-derived MP and SP cells have not been fully characterized directly ex vivo. Using flow cytometry, MP and SP cells were characterized based on their expression of several well-accepted progenitor cell antigens. Both the MP and SP populations are heterogeneous and overlapping in the cells they contain. The percentages of cells in each population vary with species and specific muscle examined. MP and SP populations contain both satellite and multipotent progenitor cells, based on expression of CD34, Sca-1, Pax7, and M-cadherin. Thus, isolation using this procedure cannot be used to predict downstream differentiation outcomes, and explains the conflicting literature on these cells. Hoechst dye also results in significant mortality of sorted cells. As defined subpopulations are easily obtained using flow cytometry, sorting immediately ex vivo based on accepted myogenic precursor cell markers will yield superior results in terms of cell homogeneity for transplantation therapy.

Purpose: Human extraocular muscles (EOM) are preferentially susceptible to thyroid eye disease. Although the specific cause of this autoimmune disorder is unknown, it is often associated with elevated thyroid hormone levels. Thus, the effect of elevated thyroid hormone levels on cross-sectional area, myofiber size, satellite cells, and myosin heavy chain (MyHC) isoform expression was examined in adult rabbit EOMs, to determine how elevated thyroid hormone alters EOM biology., Methods: After 1 month of elevated thyroid hormone levels, the EOMs were removed and prepared for histologic examination. Total muscle mass, myofiber size, patterns of MyHC isoform expression, and the number of satellite cells were determined., Results: Elevated thyroid hormone levels significantly decreased muscle mass, total number of myofibers, and mean cross-sectional area of the myofibers. Alterations in MyHC isoform expression were extremely complex, but several basic patterns emerged. The percentages of neonatal- and developmental-positive myofibers decreased in almost all EOM regions examined, and the percentages of slow-positive myofibers significantly increased. In contrast to normal EOMs, which retain a population of activated satellite cells throughout life, elevated thyroid hormone levels resulted in the virtual disappearance of MyoD-positive cells and a decrease in Pax7-positive cells., Conclusions: The reductions in EOM size, number of fibers expressing developmental and neonatal MyHC, and number of MyoD- and Pax7-positive satellite cells suggest that elevated thyroid hormone levels decrease the ongoing myofiber remodeling normally seen in the EOM. These catabolic changes have important implications for maintenance of function in the EOMs.

Purpose: The goal of this study was to determine whether the medial rectus muscles of patients with a history of medial rectus underaction or overaction show alterations in the process of satellite cell activation when compared with normal age-matched control muscles., Methods: Medial rectus muscles were obtained with consent from adult patients undergoing surgical resection due to medial rectus underaction or overaction and were prepared for histologic examination by fixation and paraffin embedding. Control muscles were obtained from cornea donor eyes of adults who had no history of strabismus or neuromuscular disease. Cross sections were obtained and stained immunohistochemically for the presence of activated satellite cells, as identified by MyoD immunoreactivity, and the presence of the total satellite cell population, as identified by Pax7 immunoreactivity. The percentages of MyoD- and Pax7-positive satellite cells per 100 myofibers in cross section were calculated., Results: As predicted from results in the literature, MyoD-positive satellite cells, indicative of activation, were present in both the control and resected muscles. In the underacting medial rectus muscles, the percentages of MyoD- and Pax7-positive satellite cells, based on the number of myofibers, were approximately twofold higher than the percentages in the control muscles. In the overacting medial rectus muscles, the percentage of MyoD-positive satellite cells was twofold less than in the control muscles, whereas the percentage of Pax7-positive satellite cells significantly increased compared with that in the control specimens., Conclusions: The presence of an increased number of activated satellite cells in the resected underacting medial rectus muscles and the decreased numbers of activated satellite cells in the overacting muscles was unexpected. The upregulation in the number of MyoD-positive satellite cells in underacting muscles suggests that there is potential for successful upregulation of size in these muscles, as the cellular machinery for muscle repair and regeneration, the satellite cells, is retained and active in patients with medial rectus underaction. The decreased number of activated satellite cells in overacting MR muscle suggests that factors as yet unknown in these overacting muscles are able to affect the number of satellite cells and/or their responsiveness compared with normal age-matched control muscles. These hypotheses are currently being tested.

Purpose: A common treatment for motility disorders of the extraocular muscles (EOMs) is a resection procedure in which there is surgical shortening of the muscle. This procedure results in rotation of the globe toward the resected muscle, increased resting tension across the agonist-antagonist pair, and stretching of the elastic components of the muscles. In the rabbit, due to orbital constraints and limited rotation, resection results in more significant stretch of the surgically treated muscle than the antagonist. This surgical preparation allows for the examination of the effects of surgical shortening of one rectus muscle and passive stretch of its ipsilateral antagonist., Methods: The insertional 6 mm of the superior rectus muscles of adult rabbits were resected and reattached to the original insertion site. After 7 and 14 days, the animals were injected intraperitoneally with bromodeoxyuridine (BrdU) every 2 hours for 12 hours, followed by a 24-hour BrdU-free period. All superior and inferior rectus muscles from both globes were examined for BrdU incorporation, MyoD expression, neonatal and developmental myosin heavy chain (MyHC) isoform expression, and myofiber cross-sectional area alterations., Results: In the resected muscle and in the passively stretched antagonist muscle, there was a dramatic increase in the number of myofibers positive for neonatal MyHC and in the number of BrdU- and MyoD-positive satellite cells. The addition of BrdU-positive myonuclei increased from 1 per 1000 myofibers in cross sections of control muscles to 2 to 3 per 100 myofibers in the resected muscles. Single myofiber reconstructions showed that multiple BrdU-positive myonuclei were added to individual myofibers. Addition of new myonuclei occurred in random locations along the myofiber length of single fibers. There was no correlation between myofibers with BrdU-positive myonuclei and neonatal MyHC isoform expression., Conclusions: Both active and passive stretch of the rectus muscles produced by strabismus surgery dramatically upregulated the processes of satellite cell activation, integration of new myonuclei into existing myofibers, and concomitant upregulation of immature myosin heavy chain isoforms. Understanding the effects of strabismus surgery on muscle cell biological reactions and myofiber remodeling may suggest new approaches for improving surgical outcomes.

Purpose: Mammalian extraocular muscles (EOMs) are both physiologically and biochemically unique when compared with nonocular skeletal muscles. Recent studies have demonstrated a process of continuous myonuclear addition in normal uninjured myofibers in adult EOMs of rabbits and mice. The current study was conducted to determine whether this process of myonuclear addition is a universal phenomenon in mammalian EOMs., Methods: The EOMs from adult uninjured monkeys and humans were examined immunohistochemically for the expression of specific markers of activated satellite cells: hepatocyte growth factor (HGF); the myogenic regulatory factors MyoD, myogenin, and Pax7; and a marker for nuclei in all proliferative phases of the cell cycle, Ki-67. The satellite cell identity of the cells positive for Ki-67, HGF, and Pax7 was determined by colabeling sets of serial sections with either laminin or dystrophin., Results: In cross sections of monkey and human EOMs, approximately 7% to 8% of the myofiber profiles were associated with Pax7-positive satellite cells and between 2% and 4% were associated with MyoD-positive satellite cells or HGF-positive satellite cells. Similar percentages of satellite cells were positive for myogenin in the orbital layer, but the global layer had few satellite cells that were myogenin positive. An average of 0.72% of the myofibers had Ki-67-positive cells associated with them in the satellite cell position., Conclusions: Activated satellite cells were present on myofibers in normal uninjured adult monkey and human EOMs, as visualized with these five distinct markers. The data support the hypothesis that the process of continuous myonuclear addition is most likely active in primate and human EOMs. The presence of continuous myofiber remodeling in EOM suggests new mechanisms that may be responsible for EOM sparing or involvement in skeletal muscle diseases.

Purpose: We recently demonstrated that there is a continuous process of myonuclear addition into normal, uninjured adult myofibers in rabbit extraocular muscles (EOM). This phenomenon is not seen in skeletal muscles from normal, adult limbs. These features may explain the selective involvement of the EOM in progressive external ophthalmoplegia and oculopharyngeal muscular dystrophy due to an accumulation of damaged DNA in mitochondria and nuclei within the EOM as a result of repeated cycling of the muscle satellite cells. Many testable hypotheses flow from these observations. We investigated whether continuous myonuclear addition is present in normal mouse EOM so that mouse models of genetic disorders can be used to study the pathogenic mechanisms and to test potential therapies for human muscle disorders., Methods: Bromodeoxyuridine (brdU) was injected intraperitoneally into C57 adult mice every 2 hours for 12 hours. Twenty-four hours later the animals were sacrificed, and the globes with the muscles attached were prepared for immunohistochemical localization of brdU-positive nuclei within the EOM. All cross sections were immunostained for both brdU and either dystrophin or laminin., Results: All the rectus muscles from the mouse EOM examined contained both satellite cells and myonuclei that were positive for brdU. This demonstrates the division of satellite cells and the fusion of their daughter cells with existing adult EOM myofibers in mice., Conclusions: These data indicate that the process of continuous myonuclear addition is also active in mouse EOM. These findings will allow various mutant mouse models to be used to study the pathogenesis and treatment of various muscle disorders. The existence of continuous myonuclear addition in adult, uninjured EOM fundamentally changes the accepted notion that EOM myofibers are postmitotic.