Satellite cells: erythropoietin treatment and endurance training

Satellite cells (SCs) play a crucial role in skeletal muscle maintenance, repair and remodelling, but the exact mechanisms of SC activation, proliferation and differentiation are not fully elucidated yet. Recent studies provide novel information on various parameters involved, including the detection of the erythropoietin receptor (EPO‐R) in cultured human primary satellite cells. Erythropoietin (EPO) is a well‐known stimulator of red blood cell production, but more interestingly, previous research has shown that EPO may also promote myoblast proliferation, survival and muscle regeneration. Current scientific evidence about the direct effects of EPO on skeletal muscle is not consistent and several differences emerge when trying to replicate the results of in vitro or animal studies on healthy humans. Furthermore, the validity of antibodies used to detect EPO‐R protein expression in several studies has been criticized due to a possible cross‐reaction between EPO‐R antibody and unspecific proteins and thus, the generation of false positive results. These issues have created uncertainty about the presence of EPO‐R in human skeletal muscle.

In a recent article published in The Journal of Physiology, Hoedt et al. (2016) examine the effects of an erythropoiesis‐stimulating agent (ESA; darbepoetin‐α), and endurance training on SC quantity and function, and investigate whether EPO‐R is expressed in freshly sorted SCs from adult human muscle. The authors hypothesized ESA treatment and endurance training would increase the SC pool and myogenic commitment and that EPO‐R would be expressed in isolated SCs. To test these hypotheses, Hoedt and colleagues assessed a number of cellular parameters in 35 healthy, untrained men, randomly divided into four similar groups. For a 10 week period each group received ESA or placebo while two of the groups also performed an endurance training protocol three times per week. Quantification of fibre type‐specific SCs (Pax7⁺), myonuclei, active SCs (Pax7⁺/MyoD⁺), differentiating SCs (Pax7^–/MyoD⁺) and indices of muscle fibre remodelling (embryonic and neonatal myosin heavy chain) were performed by immunohistochemistry using muscle samples from the vastus lateralis muscle. The detection of EPO‐R mRNA in adult SCs was performed by real‐time RT‐PCR using fluorescence‐activated cell sorting (CD56⁺/CD45^–/CD31^–) from the rectus abdominis muscle.

A considerably higher level of EPO‐R mRNA was detected in SCs compared with whole homogenized muscle, supporting the hypothesis of a direct action of EPO on skeletal muscle through EPO‐R. However, Hoedt and colleagues evaluated EPO‐R mRNA expression in only a single subject, a fact that limits the power of data interpretation and also may ignore potential inter‐individual differences. Also, despite the novel findings, the results should be interpreted with caution, as mRNA levels do not necessarily mirror protein levels. The authors did not determine EPO‐R protein, a fact which preserves the controversy on this issue. Lamon et al. (2014) reported a discrepancy between the regulation of EPO‐R mRNA and protein levels during proliferation and differentiation in human primary myoblasts (2‐fold decrease vs. 30‐fold increase, respectively). Nevertheless, no safe conclusions can be extracted from the comparison between the two studies, due to the examination of different type of cells (myoblasts vs. freshly isolated SCs). Furthermore, Christensen et al. (2015) failed to detect EPO‐R protein in human skeletal muscle despite the presence (although very low) of EPO‐R mRNA, while the same study did not find any significant gene expression variation in skeletal muscle after 10 weeks of ESA treatment. However, this result may be partly explained by a possible inadequacy or delay of ESA treatment alone in producing a sufficient gene expression response. Also, a possible prime or exclusive expression of EPO‐R in SCs would limit the probability of detecting the protein content in whole muscle homogenate. Moreover, the different anatomical locations chosen for EPO‐R muscle sampling compared with Hoedt et al. (vastus lateralis vs. rectus abdominis) might explain some differences in EPO‐R mRNA levels between the two studies that might have in turn affected EPO‐R protein expression.

Hoedt et al. (2016) is the first study demonstrating a myogenic response to exogenous EPO administration in human freshly isolated muscle. The authors detected a main effect of ESA treatment with significant increases in Pax7⁺/MyoD⁺ and MyoD⁺ cell content. These results confirm the hypothesis that ESA treatment increases the myogenic commitment of SCs and support the possibility that SCs may act as mediators of the myogenic action of EPO. Zhang et al. (2010) report decreased MyoD expression in SCs of mice with chronic kidney disease (CKD), but there is no study focusing on the effects of EPO treatment on SCs of CKD or other patients. Moreover, Hoedt and colleagues detect high EPO‐R mRNA levels in isolated CD90⁺/Lin⁻ cells, suggesting that mesenchymal stem cells may mediate the effects of EPO on SCs through inducing MyoD expression in SCs or through activating myogenic factors such as follistatin. Nevertheless, further research is needed to examine the potential role of mesenchymal cells in mediating EPO and SCs. Despite the increased MyoD expression, no significant changes have been observed in neonatal myosin heavy chain, centrally located nuclei and myonuclei content and no markers of EPO‐activated signalling cascades (e.g. JAK2/STAT5, PI3‐K/Akt) were investigated. Also, Larsen et al. (2014) found no effects of EPO treatment on skeletal muscle morphology or angiogenesis in healthy young men, supporting the debate about the myogenic effects of EPO administration on skeletal muscle. Hoedt et al. suggest that, even though EPO may stimulate myoblast proliferation via MyoD expression, it might also reduce myogenin expression and thus inhibit or postpone myoblast differentiation. Importantly, the authors state that EPO might lead to a reduction in the SC pool over a prolonged time period by enhancing the myogenic commitment process without producing a similar response in the self‐renewal mechanism. The latter conclusions are also supported by the lack of change in SC content observed after ESA treatment and the positive trend (P = 0.085) towards an ESA effect on Pax7⁻/MyoD⁺ cells.

The authors also examine the effects of endurance training independently and in combination with ESA treatment. Results show that endurance training increased SC levels in type II myofibres, in addition to MyoD expression. Nonetheless, a combined effect of ESA and endurance training on SC quantity or myogenic cell activation was not detected. ESA treatment, however, did not negatively affect the increase in SC content induced by endurance training, and also there were no increases in MyoD⁺, Pax7⁺/MyoD⁺ cell levels and the indices of muscle fibre remodelling resulted from the combination of the two interventions. Although EPO may concur with endurance training to improve aerobic capacity and performance, these findings suggest that it may even interfere with some skeletal muscle adaptations to exercise in previously sedentary individuals. Furthermore, this interference seems to alter MyoD expression and therefore affect the myoblast commitment, although this might not be the only involved mechanism. Whether or not these effects would be observed in a longer term in patients and/or in populations that have already developed chronic muscle adaptations (i.e. endurance athletes) needs to be clarified.

To conclude, Hoedt et al. (2016) provide some interesting novel findings regarding the effects of EPO on MyoD expression in SCs, the presence of EPO‐R mRNA in freshly isolated human primary SCs and the effects of ESA treatment on mediating the myogenic response after endurance training. However, no definitive conclusion on the function of EPO‐R on SCs can be drawn from this study due to the lack of EPO‐R protein detection. Future studies would expand our knowledge about the effects of EPO on SCs and the role of EPO‐R in SC proliferation and differentiation. Further research is needed to examine the possible benefits from EPO treatment in SCs of patient populations and the physiological interplay between EPO and different types of training stimuli. In this last regard, and since EPO is physiologically provoked by hypoxia while endurance training improves aerobic capacity, an interesting objective for future studies would be to examine the combination of ESA treatment with hypoxic or resistance training on SCs and muscle morphology.

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Competing interests

None declared.

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