Identification and characterization of cellular and molecular mechanisms responsible for the regulation of skeletal muscle mass during experimental hyperthyroidism.

Thyroid hormones are essential players on cellular singling, primarily through the regulation of gene transcription by triiodothyronine (T3). However, supraphysiological levels of T3, also called hyperthyroidism, leads to pathological changes in target tissues. In skeletal muscle, hyperthyroidism is related to atrophy throughout mechanisms that may include proteolytic stimulation and inhibition of protein synthesis. To get new insights about T3 action upon skeletal muscle, we sought to identify new regulation targets by analyzing the global expression of genes involved on both protein synthesis and degradation pathways. Regarding the catabolic axis, we identified upregulation of the MDM2 E3 ligase exclusively in fast twitch fibers. Additionally, MDM2 is found in the cytoplasm and nucleus, in the latter colocalized with the transcription factor PAX7. In response to T3, there is augmented MDM2 nuclear translocation and reduced PAX7 colocalization. Similarly, T3 inhibits FOXO3 nuclear translocation suggesting a MDM2- dependent inactivation mechanism. Furthermore, pharmacological MDM2 inhibition in cell culture intensified T3 effects, resulting smaller myotubes with high atrogenes expression. Thus, our results indicate that MDM2 may integrate a protrophic response to T3 in skeletal muscle. On the other hand, the analysis of mTOR components showed a swift and strong downregulation of Rptor, a fundamental subunit of mTORC1 complex. Surprisingly, we did not find T3-responsive elements in the promoter region of Rptor, so we explored the hypothesis of indirect inhibition of Rptor by T3 through the myostatin pathway. In fact, we verified myostatin positive response to T3. However, we identified inhibited SMAD3 nuclear translocation. Furthermore, T3 level levels enhanced de novo protein synthesis despite the low phosphorylation of mTOR and P70S6K. Finally, myostatin silencing or Rptor ectopic expression protected skeletal muscle from T3-induced atrophy. Thus, we conclude that Rptor inhibition is essential for the establishment of T3-induced atrophy, which in turn takes place regardless of the levels of protein synthesis levels. (AU)