Myokines as potential indirect biomarkers of myostatin abuse in sport doping: reference ranges in elite athletes
Authors: Donati Francesco , Biasini Giorgia Morgan , de la Torre Xavier , Lasalvia Alba , Noce Francesco , Rutigliano Lavinia , Sian Veronica , Botre Francesco 
Published: April 2020

Background

Myokines are a class of small proteins mainly produced and released from muscle cells and involved in muscle contractions, exercise‐associated metabolic changes and myogenesis. Among them, myostatin (MYO) is one of the key negative regulators of skeletal muscle cell growth and differentiation. As a consequence, the abuse of myostatin inhibitors as performance‐enhancing agents in sport, and/or the use of pharmaceutical agents or genetic methods blocking myostatin function are presently prohibited by the World Anti‐doping Agency (WADA). No direct method is currently available for the detection of all the many possible strategies of myostatin inhibition. We are here exploring the potential application of human serum myokines as indirect markers of doping. We are here reporting the results of a pilot study aimed to asses baseline serum levels of the principal myokines, and their variability as a function of sex, age and sport discipline.

Methods

We have assayed MYO in human serum, together with a variety of myokines: follistatin‐like 1 (FSTL‐1), musclin (MUS), oncostatin (ONC), osteonectin (OST), irisin (IRI), brain‐derived neurotrophic factor (BDNF), some interleukins (IL‐6 and IL‐15) and the main inhibitor of the members of the TGF‐β superfamily follistatin (FST). Serum samples from elite athletes were assayed through various immunological assays using antibodies specifically directed against the proteins of interest. More specifically, MYO and FST levels were measured by enzyme‐linked immunosorbent assay (ELISA); IRI serum levels by radioimmunoassays (RIA), while the analysis of the other peptides by multiplex cytometric beads array (CBA).

Results

All analytes resulted measurable in human serum, with the exception of few samples relatively to MUS and IRI. The myokines analyzed showed levels of serum concentration of different order in magnitude: OST and MUS had a concentration lower than 1ng/mL; FST, MYO and IRI reached very few ng/mL of concentration, while BDNF and FSTL‐1 were highly expressed in serum (10–50 ng/mL). No significant differences in myokines serum levels were found between female and male subjects. Our results showed significantly lower levels of FST in subjects under the age of 25 compared to the older ones, but no differences were found for the other proteins. Interestingly the ratio between MYO and FST is higher in the great majority of all the sample analyzed.

Conclusions

In this pilot study we examined a pool of human myokines to assess their variability in serum samples of athletes and their potential feasibility as biomarkers to reveal the manipulation of muscle cells regulators for doping purpose. In general, there were no strong correlations among the myokines studied, despite a PCA analysis showed that a group of 4 myokines (MUS, FSTL‐1, ONC, MYO) were more closely related. These preliminary results suggest that the identification of a range of baseline levels, especially for these 4 myokines, and their longitudinal monitoring, may represent an innovative strategy to reveal the illicit recourse to doping strategies aimed to myostatin inhibition.

Support or Funding Information

Federazione Medico Sportiva Italiana