Myostatin Inhibition Attenuates T3‐induced Skeletal Muscle Atrophy
Authors: Cruz André , da Silva William Jose , Moriscot Anselmo Sigari 
Published: April 2020

Support or Funding Information

São Paulo Research Foundation (FAPESP) Grants #2017/09398‐8, and #2015/04090‐0.

Skeletal muscle is a major tissue accountable for many key functions such as breathing, thermogenesis, whole body movement and aminoacid source. In addition, skeletal muscle is able to change both its size and force according to variations in mechanical demands and internal conditions such as hormonal levels, notably corticoids and triiodothyronine (T3). Nevertheless, pathological rise in T3 levels results in skeletal muscle atrophy. Although it has been reported that increased proteolysis is involved, the role of other biological processes, such as protein synthesis, in skeletal muscle T3 action, are not well understood. Thus we performed a microarray analysis from skeletal muscles of hyperthyroid rats focusing on protein synthesis related mRNAs. We observed a consistent inhibition in raptor expression, suggesting disturbance of mTORC1 signaling; however, the absence of T3 responsive elements in raptor promoter region indicates that an intermediate gene could be targeted by the hormone. We then searched the literature for prospects and myostatin caught our attention: its expression is induced by T3 via TREs, has a clear inhibitory effect upon mTORC1, and a possible regulatory feedback with raptor. Thus, we hypothesized that T3 can increase myostatin expression, which in turn inhibits raptor expression, destabilizing mTORC1 and consequentely impairing protein synthesis, leading muscle atrophy. To start addressing this hypothesis, we investigated the role of myostatin in T3‐induced skeletal muscle atrophy. Experimental hyperthyroidism was induced in Wistar rats (~260g) by T3 daily doses (6μg/100g) for 7 and 14 days, animals were killed, and soleus muscle collected. Electroporation was utilized to induce miRNA expression in rat soleus muscle. All animal procedures were approved under the ethics committee #04/2016. As expected T3 induced decreased Fiber cross‐sectional (~40% at 7 and 14 days) and fiber type twitch. Myostatin expression was up‐regulated at 7th and 14th day (~3 and ~2 fold, respectively) while raptor was down‐regulated after 14 days of treatment (0.6 fold). In addition, T3 reduced p‐mTOR, p‐P70S6k, and p‐4EBP1 (0.5, 0.3, 0.4 fold respectively). Intriguily, de novo protein synthesis increased after T3 treatment both in vivo (7 day 2.5 fold; 14 days 1.7 fold) and in vitro (C2C12 72h – 1.5 fold). Finally, we inhibited myostatin expression in vitro using interference RNA and observed that myostation inhibition blocked the decrease in diameter induced by T3 and restored raptor expression. In order to get further insight into molecular mechanisms, we searched for miRNAs responsive to T3 with validated myostatin inhibition and miR‐208a caught our attention. We forced miR‐208a expression in soleus from rats under experimental hyperthyroidism for 7 days and observed that miR‐208a expression was capable of attenuating T3‐induced decrease in soleus muscle cross‐sectional area (~46% T3 vs control; ~14% T3+miR208a vs control). In conclusion, our results indicate that myostatin is a important component of T3 atrophic stimulus in skeletal muscle.