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Randomized Controlled Trial
. 2016 Feb 1;594(3):727-43.
doi: 10.1113/JP271333. Epub 2015 Dec 30.

Satellite Cell Response to Erythropoietin Treatment and Endurance Training in Healthy Young Men

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Free PMC article
Randomized Controlled Trial

Satellite Cell Response to Erythropoietin Treatment and Endurance Training in Healthy Young Men

Andrea Hoedt et al. J Physiol. .
Free PMC article

Abstract

Erythropoietin (Epo) treatment may induce myogenic differentiation factor (MyoD) expression and prevent apoptosis in satellite cells (SCs) in murine and in vitro models. Endurance training stimulates SC proliferation in vivo in murine and human skeletal muscle. In the present study, we show, in human skeletal muscle, that treatment with an Epo-stimulating agent (darbepoetin-α) in vivo increases the content of MyoD(+) SCs in healthy young men. Moreover, we report that Epo receptor mRNA is expressed in adult human SCs, suggesting that Epo may directly target SCs through ligand-receptor interaction. Moreover, endurance training, but not Epo treatment, increases the SC content in type II myofibres, as well as the content of MyoD(+) SCs. Collectively, our results suggest that Epo treatment can regulate human SCs in vivo, supported by Epo receptor mRNA expression in human SCs. In effect, long-term Epo treatment during disease conditions involving anaemia may impact SCs and warrants further investigation. Satellite cell (SC) proliferation is observed following erythropoitin treatment in vitro in murine myoblasts and endurance training in vivo in human skeletal muscle. The present study aimed to investigate the effects of prolonged erythropoiesis-stimulating agent (ESA; darbepoetin-α) treatment and endurance training, separately and combined, on SC quantity and commitment in human skeletal muscle. Thirty-five healthy, untrained men were randomized into four groups: sedentary-placebo (SP, n = 9), sedentary-ESA (SE, n = 9), training-placebo (TP, n = 9) or training-ESA (TE, n = 8). ESA/placebo was injected once weekly and training consisted of ergometer cycling three times a week for 10 weeks. Prior to and following the intervention period, blood samples and muscle biopsies were obtained and maximal oxygen uptake (V̇O2, max) was measured. Immunohistochemical analyses were used to quantify fibre type specific SCs (Pax7(+)), myonuclei and active SCs (Pax7(+)/MyoD(+)). ESA treatment led to elevated haematocrit, whereas endurance training increased V̇O2, max. Endurance training led to an increase in SCs associated with type II fibres (P < 0.05), whereas type I fibres showed no changes. Both ESA treatment and endurance training increased Pax7(+)/MyoD(+) cells, whereas only ESA treatment increased the total content of MyoD(+) cells. Epo-R mRNA presence in adult SC was tested with real-time RT-PCR using fluorescence-activated cell sorting (CD56(+)/CD45(-)/CD31(-)) to isolate cells from a human rectus abdominis muscle and was found to be considerably higher than in whole muscle. In conclusion, endurance training and ESA treatment may separately stimulate SC commitment to the myogenic program. Furthermore, ESA-treatment may alter SC activity by direct interaction with the Epo-R expressed on SCs.

Figures

Figure 1
Figure 1. Fibre type specific Pax7+ cell identification
Representative muscle cross‐section stained for Pax7 (red, A), nuclei (DAPI) and MHC‐II (blue, B), MHC‐I and laminin (green, C) and merged in (D). Three Pax7+ cells are identified (cones) and all of them associated with type I fibres (MHC‐I+). Scale bar = 50 μm.
Figure 2
Figure 2. Satellite cell content pre‐ and post‐intervention period
Satellite cells (SCs) associated with type I (A), type II (B) or hybrid (C) fibres pre and post 10 weeks of sedentary placebo (SP), sedentary ESA treatment (SE), training placebo (TP) or training ESA treatment (TE). One double positive (MHC‐I+/MHC‐II+) fibre is shown in (D) (§). SC content was expressed per fibre and data presented as individual values and the group mean ± SE (A and B) or individual values and medians (C). C, the number of fibres counted for each data point is shown next to the individual data point. Differences between pre and post are denoted by an asterisk (*P < 0.05). Group difference is denoted by a hash symbol (#P < 0.05).
Figure 3
Figure 3. MyoD+ satellite cell identification pre‐ and post‐intervention period
Representative muscle cross‐section stained for Pax7 (red, A), MyoD (green, B) and nuclei (DAPI, blue, C). One Pax7+/Myod+ cell (white cone) and one Pax7+/MyoD (yellow cone) are identified. Scale bar = 50 μm. Quantification of MyoD+ cells (D), MyoD+/Pax7+ cells (E) and MyoD+/Pax7 cells (F) pre and post 10 weeks of sedentary placebo (SP), sedentary ESA treatment (SE), training placebo (TP) or training ESA treatment (TE). Cell content is expressed per fibre and data are presented as individual values and medians. Differences between pre and post are denoted by an asterisk (*P < 0.05). Group difference is denoted by a hash symbol (#P < 0.05).
Figure 4
Figure 4. Indices of fibre remodelling or regeneration pre‐ and post‐intervention period
Central nuclei identified in type I (A) or type II (B) fibres, as well as fibres expressing neonatal (n)MHC (C) pre and post 10 weeks of sedentary placebo (SP), sedentary ESA treatment (SE), training placebo (TP) or training ESA treatment (TE). Two nMHC+ fibres (green) are shown in (D) with basal lamina delineated by laminin (red). Central nuclei content or nMHC+ fibres are expressed per fibre and data are presented as individual values and medians.
Figure 5
Figure 5. Epo‐R mRNA expression in adult human satellite cells
AC, contour plots indicating the FACS strategy are shown. Three cell populations were identified; CD56+ cells (satellite cells) [CD56+/Lin (CD45/CD31)], CD90+ cells (CD90+/Lin) and Lin+ (CD45+/CD31+). As shown in (C), the CD56+ and CD90+ cell populations were largely negative for CD90 and CD56, respectively. In addition to FACS sorted cells, Epo‐R expression was determined in whole muscle homogenate (MH) and a pre‐FACS sample. mRNA expression levels of Epo‐R (D) were quantified and normalized to mRNA expression of ribosomal protein large P0 (RPLP0). Each target was expressed relative to whole muscle homogenate (MH). Data (n = 1) are shown on a log2 scale.

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