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. 2011 Apr 1;317(6):873-85.
doi: 10.1016/j.yexcr.2011.01.018. Epub 2011 Jan 27.

Sparing of extraocular muscle in aging and muscular dystrophies: a myogenic precursor cell hypothesis

Kristen M Kallestad  1 , Sadie L Hebert, Abby A McDonald, Mark L Daniel, Sharon R Cu, Linda K McLoon
Affiliations

Sparing of extraocular muscle in aging and muscular dystrophies: a myogenic precursor cell hypothesis

Kristen M Kallestad et al. Exp Cell Res. .

Abstract

The extraocular muscles (EOM) are spared from pathology in aging and many forms of muscular dystrophy. Despite many studies, this sparing remains an enigma. The EOM have a distinct embryonic lineage compared to somite-derived muscles, and we have shown that they continuously remodel throughout life, maintaining a population of activated satellite cells even in aging. These data suggested the hypothesis that there is a population of myogenic precursor cells (mpcs) in EOM that is different from those in limb, with either elevated numbers of stem cells and/or mpcs with superior proliferative capacity compared to mpcs in limb. Using flow cytometry, EOM and limb muscle mononuclear cells were compared, and a number of differences were seen. Using two different cell isolation methods, EOM have significantly more mpcs per mg muscle than limb skeletal muscle. One specific subpopulation significantly increased in EOM compared to limb was positive for CD34 and negative for Sca-1, M-cadherin, CD31, and CD45. We named these the EOMCD34 cells. Similar percentages of EOMCD34 cells were present in both newborn EOM and limb muscle. They were retained in aged EOM, whereas the population decreased significantly in adult limb muscle and were extremely scarce in aged limb muscle. Most importantly, the percentage of EOMCD34 cells was elevated in the EOM from both the mdx and the mdx/utrophin(-/-) (DKO) mouse models of DMD and extremely scarce in the limb muscles of these mice. In vitro, the EOMCD34 cells had myogenic potential, forming myotubes in differentiation media. After determining a media better able to induce proliferation in these cells, a fusion index was calculated. The cells isolated from EOM had a 40% higher fusion index compared to the same cells isolated from limb muscle. The EOMCD34 cells were resistant to both oxidative stress and mechanical injury. These data support our hypothesis that the EOM may be spared in aging and in muscular dystrophies due to a subpopulation of mpcs, the EOMCD34 cells, that are retained in significantly higher percentages in normal, mdx and DKO mice EOM, appear to be resistant to elevated levels of oxidative stress and toxins, and actively proliferate throughout life. Current studies are focused on further defining the EOMCD34 cell subtype molecularly, with the hopes that this may shed light on a cell type with potential therapeutic use in patients with sarcopenia, cachexia, or muscular dystrophy.

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Figures

Figure 1
Figure 1
A. Percentage of all freshly isolated live mononuclear cells from wild type EOM (left) and hindlimb muscles examined for expression of M-cadherin. No significant difference in the percentage that were positive was seen. B. Percentage of all freshly isolated live mononuclear cells from wild type EOM (left) and hindlimb muscles (right) examined for expression of Pax-7. No significant difference in the percentage that were positive was seen. C. Percentage of all freshly isolated live mononuclear cells from wild type EOM and hindlimb muscles examined for expression of SM-C/2.6 (N=4) [18]. The percentage of mononuclear cells positive for this differentiated satellite cell marker was significantly greater in hindlimb than in EOM.
Figure 2
Figure 2
A. Percentage of all freshly isolated live mononuclear cells from wild type EOM and hindlimb muscles positive for Sca-1 (N=6), CD34 (N=8), M-cadherin (N=4), and Pax-7 (N=4). B. Live cells per mg of wild type EOM and hindlimb muscles positive for Sca1, CD34, M-cadherin, and Pax-7. * indicates significantly different from EOM muscle for the identical marker. C. Identification of a population of CD34+/Sca-1− significantly increased in wild type EOM compared with limb skeletal muscle. Top dot plots represent isotype control staining. Lower plots show gates for CD34+/Sca-1− and CD34+/Sca-1+ cells. D. Histograms show M-cadherin staining of each gated population; black histograms are CD34+/Sca-1− cells, white histograms are CD34+/Sca-1+ cells. E. Isotype control (white) and CD34 (black) staining of wild type EOM or hindlimb mononuclear cells, and the line indicates CD34+ gate for bottom dot plots. F. Dot plots show the Sca-1 and CD31 expression for the CD34+ cells.
Figure 3
Figure 3
A. Percentage of all freshly isolated live mononuclear cells from wild type EOM and tibialis anterior muscles (N=4). B. Percentage of all freshly isolated live mononuclear cells from wild type EOM and tibialis anterior muscles positive for Sca-1 or CD34 (N=4).
Figure 4
Figure 4
Quantification of percentage of EOMCD34 cells from EOM and hindlimb muscles of neonatal, adult and aged wild type mice (N=4). Cells were first gated on live CD31−/CD45− /Sca1− FACS dot plots. * indicates significantly different from EOM percentages. # indicates significantly different from controls based on age of animals.
Figure 5
Figure 5
Quantification of EOMCD34 cells per mg of muscle from EOM and hindlimb of wild type, mdx, and DKO mice (N=4). Cells were first gated on live, CD31−/CD45−/ Sca1− FACS dot plots.
Figure 6
Figure 6
A. EOMCD34 cells cultured on plastic culture plates in myoblast growth media for six weeks. B. Differentiation on plastic culture plates of EOMCD34 cells cultured for six weeks in proliferation media and one week in differentiation medium. C. Differentiated myotubes from EOMCD34 cells grown on glass coverslips and immunostained for the presence of desmin.
Figure 7
Figure 7
Wild type and mdx mice were treated with brdU for 28 days. After a brdU-free period of 14 days, cells were analyzed by flow cytometry for expression of CD34 and Sca-1 and subsequently for brdU. Cells were first gated on live CD31−/CD45− FACS dot plots (N=3). ANOVA was performed following angular transformation, and * indicate significant difference due to mouse phenotype and # indicates significant difference due to cell type.
Figure 8
Figure 8
Day 5 CD34+/Sca1−/CD45−/CD31− cells from (A) EOM or (B) tibialis anterior (TA) and immunostained for desmin (brown) and nuclei (purple). C. Quantification of the fusion index calculated as the number of nuclei present in myotubes divided by the total number of nuclei (N=3).
Figure 9
Figure 9
Photomicrographs of limb muscle and EOM immunostained for the presence of CD34-positive cells (blue/purple) and dystrophin (brown). A. The CD34-positive cells (black arrows) were small and irregularly located within EOM cross-sections. Two types of CD34-positive cells were present, one small and found within the perimysium (vertical arrow) and one larger, found adjacent to the dystrophin-positive sarcolemma (horizontal arrow). Bar equals 20 microns. B. The density of CD34+ cells was greater in the orbital layers of the EOM (orb). Within the global layer (glob), large areas were often relatively devoid of cells. C. Blood vessel in the EOM devoid of CD34-positive cells (green arrow). Note positive cell cluster around a single myofiber (black arrow). D. The nerve was also devoid of CD34-positive cells (green asterisks). Black arrow indicates a CD34-positive cell in the satellite cell position. E. Section through a leg muscle cutting a blood vessel tangentially and immunostained for CD34. CD34+ cells in the satellite cell position were rare in leg muscle. They were usually associated with blood vessels. Inset is a higher power version of positive cells found associated with the blood vessel (black arrow). Magnification bars equal 10 microns.
Figure 10
Figure 10
A. FACS dot plots show the forward-side scatter (top) and CD34/Sca-1 expression (bottom) gating strategies of mononuclear cells from muscle. Histograms show the percentage of cells that are 7AAD+ based on FACS gating that included CD34+/Sca-1+ cells (top 3), or CD34+/Sca-1− (bottom 3). B. Percentages of dead or dying cells from EOM and limb skeletal muscle of wild type, mdx, and DKO animals are indicated (N= 3). C. Percentage of apoptotic cells defined by Annexin-V and CD34+staining of cultured wild type mononuclear cells 24 hours after treatment with H2O2 (N=5).
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