A cellular memory mechanism aids overload hypertrophy in muscle long after an episodic exposure to anabolic steroids

Abstract

Previous strength training with or without the use of anabolic steroids facilitates subsequent re-acquisition of muscle mass even after long intervening periods of inactivity. Based on in vivo and ex vivo microscopy we here propose a cellular memory mechanism residing in the muscle cells. Female mice were treated with testosterone propionate for 14 days, inducing a 66% increase in the number of myonuclei and a 77% increase in fibre cross-sectional area. Three weeks after removing the drug, fibre size was decreased to the same level as in sham treated animals, but the number of nuclei remained elevated for at least 3 months (>10% of the mouse lifespan). At this time, when the myonuclei-rich muscles were exposed to overload-exercise for 6 days, the fibre cross-sectional area increased by 31% while control muscles did not grow significantly. We suggest that the lasting, elevated number of myonuclei constitutes a cellular memory facilitating subsequent muscle overload hypertrophy. Our findings might have consequences for the exclusion time of doping offenders. Since the ability to generate new myonuclei is impaired in the elderly our data also invites speculation that it might be beneficial to perform strength training when young in order to benefit in senescence.

Figure 1. Effect of anabolic steroids and functional overload on cross-sectional area (CSA) and the number of myonuclei per fibre found per fibre on cross-sections (CS)

A, representative micrographs of cryosections stained with Hoechst dye 33342 (blue) to label DNA, and antibodies against dystrophin (green) from soleus muscles after 2 weeks of sham or steroid treatment and with or without simultaneous functional overload. Scale bar is 100 μm. CSA, and number of myonuclei per fibre cross-section (CS) from soleus (B and D), and EDL (C and E). Bars show means ± SEM. Letters a–d above columns indicate statistical significant differences (P < 0.01); columns designated with the same letter were not statistically different. Each column represents 317–896 fibres from 6 muscles.

Figure 2. The effect of prior administration of anabolic steroids on the number of nuclei myonuclei per fibre found on cross-sections (CS; A) and fibre cross-sectional area (CSA; B)

Inset in A shows blood testosterone concentration on same time axis as in main figure. *Statistically significantly different from sham (P < 0.05); #statistically significantly different from overload (P < 0.05). Each data point represents 300 fibres from 6 muscles. C, representative micrographs of cryosections stained with Hoechst dye 33342 (blue) to label DNA, and antibodies against dystrophin (green) after treatment with anabolic steroid or sham pellets for 2 weeks, 3 weeks after pellet removal, and followed by functional overload for 2 weeks. Scale bar is 50 μm.

Figure 3. In vivo imaging demonstrated that prior administration of anabolic steroids increased the number of myonuclei per mm fibre (A and B) and enhanced a subsequent hypertrophy response to overload in EDL muscles (A and C)

*Statistically significantly different from sham (P < 0.001); #statistically significantly different from overload (P < 0.001). Each data point represents 33–50 fibres from 6–8 muscles. C, representative in vivo images of muscles each showing three oligo-injected muscle fibres labelling myonuclei and the outline of single fibres immediately after treatment with anabolic steroid or sham pellets for 2 weeks, 3 weeks after pellet removal, and followed by functional overload for 2 weeks. In A hairlines indicate boundaries when different focal planes are depicted. Scale bar is 50 μm.

Figure 4. Long-term effects of a short period of anabolic steroid administration on myonuclei (A) and fibre growth in response to overload in EDL muscles (B and C)

*Statistically significantly different from sham (P < 0.001); #statistically significantly different from overload (P < 0.001). Each data point represents 669–856 fibres from 6 muscles. C, representative micrographs of cryosections stained with Hoechst dye 33342 (blue) to label DNA, and antibodies against dystrophin (green) immediately after treatment with anabolic steroids or sham pellets for 2 weeks and muscles that 3 months later were functionally overloaded for 6 and 14 days. Scale bar is 300 μm.