Implications of RNA-seq in the detection of anabolic steroid use and harnessing of the molecular mechanism of muscle memory

Code: ISF16E11FP 

The variable effects of anabolic androgenic steroids (AAS) on increasing skeletal muscle mass and strength has been well documented, as is the misuse of AAS in sport. Recent evidence suggest either a long- or short-term exposure to AAS might have a sustained effect on muscle morphological changes, for example, with increased muscle mass, capillary per fibre, muscle fibre size and myonuclei density, leading to improved performance. A positive correlation between the number of myonuclei and training response
following exposure to AAS in a mouse model seems to suggest a link between the formation of extra myonuclei and the extent of “muscle memory”; an idea that requires further investigation in humans.

Strength training can also increase the number of nuclei in muscle fibres. Adaptations in muscle mass by strength training are significantly enhanced in previously trained individuals despite a prolonged detraining period. Given the persistence of muscle nuclei, the use of AAS combined with training will have a greater impact on muscle hypertrophy than either training or steroid use alone. To detect the long-term effect of AAS and training (even after drugs are no longer detectable in the human system), abnormal changes in
skeletal muscle morphology illustrated by specific gene markers in response to the stimuli exist and will allow these molecular signals to be picked up by modern gene screening methods. In the proposed project, gene expression profiling of skeletal muscle in response to AAS exposure will be carried out using total RNA-seq. The molecular, histological and training response markers will be integrated for the detection of short- and long-term effects of AAS and will be incorporated into the steroid module of the Athlete Biological
Passport for improved validity and reliability.
 

Main Findings:

Fat Free Mass (FFM) of RP2-5, who ceased AAS usage ≤2 weeks prior to visit one with 19-28 weeks between visits, decreased by 3.9-4.7 kg. FFM of RP1, who ceased AAS usage 34 weeks prior to visit one with 28 weeks between visits, decreased by 0.9 kg. Fibre CSA decreased for RP1 and RP2 between visits (7566 vs 6629 µm²; 7854 vs 5677 µm²) whilst myonuclei per fibre remained similar (3.5 vs 3.4; 2.5 vs 2.6). Fibre CSA (7167 vs 7889 µm²) and myonuclei per fibre (2.6 vs 3.3) increased for RP3 between visits. Mean fibre CSA was significantly higher in RT-AS (n=17) (8160 ± 1769 µm²) compared to C (n=5) (6477 ± 1271 µm², p=0.028). There were no significant differences between C, RT (n=15), RT-AS & PREV (n=6) for myonuclei per fibre. Myonuclei per fibre and CSA for all biopsied participants (n=43) was significantly correlated (r=0.8, p<0.001). 
All whole blood samples (n=60) were subjected to RNA-Seq as they had purified total RNA that was of sufficient concentration, purity, and integrity. In comparison with RT participants who ceased AAS exposure <1 week ago (n=10), 1-2 weeks ago (n=5), 10-50 weeks ago (n=4), and >52 weeks ago (n=7), had 612, 464, 173 and 188 genes differentially expressed, respectively. RP2-5 had 33 differentially expressed genes between visits which were mainly associated with the interferon signalling pathway linked to the immune system. RNA-Seq of corresponding muscle samples is currently underway. The finding of comparable myonuclei per fibre numbers despite decrements in fibre CSA post AAS usage is consistent with the “muscle memory” mechanism. RNA-Seq identified 33 genes that could provide novel beneficial biomarkers. 
Further research, particularly the longitudinal monitoring of AAS users post usage is required to confirm these intriguing findings.