The extraocular muscles (EOM) are spared in Duchenne muscular dystrophy patients (DMD) and continue to function after most skeletal muscles in the body have completely degenerated. The reason for this sparing in DMD is unknown. Unlike limb skeletal muscle, normal adult EOM retain a population of activated satellite cells, the regenerative cell in adult skeletal muscle. Satellite cells actively fuse into normal myofibers in the EOM throughout life, even in aging EOM, resulting in a continuous process of myofiber remodeling. The EOM and their satellite cells are extremely resilient to injury, denervation, disease, and aging, retaining normal morphology when limb muscle would normally atrophy. In addition, the early genes controlling EOM development are distinct from those that control somite development. We will test the hypothesis that this continuous remodeling is the process by which EOM are spared in DMD. We will examine 1) the rate of myogenic precursor cell turnover in two mouse models of DMD, the mdx and mdx/utrophin+/- (mdx/utrhet) mice, and 2) test whether inhibition of cell division in the EOM prevent their sparing in the two mouse models for DMD. The sparing of the EOM in DMD, aging, and injury and differences in the myogenic precursor cell populations in skeletal muscle suggest myogenic precursor cells (mpcs) within EOM may be significantly enriched or phenotypically distinct compared with adult limb muscle cells. There are 5-8 fold more myogenic precursor cells in adult EOM compared to limb. Additionally, mpcs from EOM are more resistant to apoptosis than similar cells from limb. We will test the hypothesis that continuous remodeling allows for EOM sparing in DMD by examining 1) the rate of myogenic precursor cell turnover in two mouse DMD models, the mdx and mdx/utrophin+/- heterozygote (mdx/utrhet) mice, and 2) whether inhibition of cell division by gamma irradiation of the EOM prevents sparing in the DMD models. Sparing of the EOM in DMD and differences in their mpcs suggest that the EOM mpcs may be significantly enriched or phenotypically distinct compared with adult limb mpcs. One population is increased;these cells are CD34+ and negative for Sca1, CD31, an endothelial lineage marker, CD45, an hematopoietic lineage marker, and negative for various satellite cell markers (EOMCD34). These EOMCD34 cells are present in the EOM and limb muscles of neonatal mice, but only maintained throughout adulthood in EOM. These cells are also present in the EOM of mdx and mdx/utrophin-/- mice. Our hypothesis is that this population of EOMCD34 cells enriched in adult EOM may be, at least in part, responsible for the sparing of EOM in DMD. We will test these hypotheses in two Specific Aims.
Specific aim 1 asks: 1) is the rate of myofiber remodeling increased in the DMD mice models? 2) Does inhibition of cell division in adult EOM prevent sparing in the EOM of mdx/utrophin +/- heterozygote mice? We will isolate specific subpopulations of mononucleated cells from EOM and limb muscles and in specific aim 2 ask: 1) Are the mpcs more resistant to injury?2) Are the mpcs from EOM more multipotent than those from limb muscle? 3) Do the myogenic precursor cells from EOM have greater proliferative potential than those derived from limb skeletal muscle? The long term goal is to define and isolate a myogenic precursor cell type from EOM that has greater proliferative and survival potential compared with limb. These cells would be tested in a myoblast transfer model to determine their potential for use in the treatment of DMD. These may offer advantages over other cells, as autologous transplants would be possible.

Public Health Relevance

The extraocular muscles (EOM) are spared in Duchenne muscular dystrophy (DMD), and the cause of this sparing is unknown. The unique ability of EOM to continuously remodel throughout life suggests that this ability may be responsible for this sparing. This may be due to an enriched myogenic precursor cell population within the EOM that has a greater ability to survive injury, aging and disease. While the eye muscles are spared in DMD, the rate of regeneration in the EOM is similar to that in the leg muscles of the mdx mouse model of DMD. We will attempt to demonstrate that muscle precursor cell division is responsible for sparing of the EOM by using irradiation to inhibit cell division. It is well known that the EOM survive injury better than limb muscle. This in turn suggests that muscle progenitors in EOM may be more robust and long-lived than those from limb muscles. If these hypotheses are true, ultimately we hope to exploit this by using identified myogenic precursor cells from EOM as a new source of donor cells in myoblast therapy in mice models of muscle injury and DMD.

National Institute of Health (NIH)
National Eye Institute (NEI)
Exploratory/Developmental Grants (R21)
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Skeletal Muscle and Exercise Physiology Study Section (SMEP)
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Araj, Houmam H
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University of Minnesota Twin Cities
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Verma, Mayank; Fitzpatrick, Krysta; McLoon, Linda K (2017) Extraocular Muscle Repair and Regeneration. Curr Ophthalmol Rep 5:207-215
McDonald, Abby A; Hebert, Sadie L; McLoon, Linda K (2015) Sparing of the extraocular muscles in mdx mice with absent or reduced utrophin expression: A life span analysis. Neuromuscul Disord 25:873-87
McLoon, Linda K; Harandi, Vahid M; Brännström, Thomas et al. (2014) Wnt and extraocular muscle sparing in amyotrophic lateral sclerosis. Invest Ophthalmol Vis Sci 55:5482-96
Kallestad, Kristen M; Hebert, Sadie L; McDonald, Abby A et al. (2011) Sparing of extraocular muscle in aging and muscular dystrophies: a myogenic precursor cell hypothesis. Exp Cell Res 317:873-85
Kallestad, Kristen M; McLoon, Linda K (2010) Defining the heterogeneity of skeletal muscle-derived side and main population cells isolated immediately ex vivo. J Cell Physiol 222:676-84
McLoon, Linda K (2009) A new role for satellite cells: control of reinnervation after muscle injury by semaphorin 3A. Focus on ""Possible implication of satellite cells in regenerative motoneuritogenesis: HGF upregulates neural chemorepellent Sema3A during myogenic differentiat Am J Physiol Cell Physiol 297:C227-30