Transcriptional regulation of ribosome biogenesis in skeletal muscle growth

Sammanfattning: Resistance exercise induces skeletal muscle hypertrophy via repeated exercise bouts. The increased muscle protein synthesis together with altered muscle protein degradation contribute to the adaptation of muscle mass. The number of ribosomes dictates the protein synthetic capacity of muscle. Therefore, the regulation of de novo ribosome synthesis is of great value for maintaining skeletal muscle mass and muscle hypertrophy. The main objective of this thesis is to examine the molecular mechanisms regulating ribosome biogenesis in response to different stimuli. In Paper I, we investigated skeletal muscle gene expression following acute and chronic resistance exercise. By examining the phosphorylation of rpS6Ser235/236 as a marker for mTOR signaling pathway activated, we found that mTOR was activated after acute resistance exercise but attenuated after 12 weeks of resistance exercise. Similar to mTOR pathway, the 45S pre-rRNA and gene expression of c-Myc showed a dramatic increase after acute resistance exercise but reduced in response to acute resistance exercise following 12 week of resistance exercise. These findings suggest that the hypertrophy associated gene expression profile is not correlated with the developing muscle phenotype. In Paper II, we exposed mice to 10% CO2 and 21% O2 in order to induce a hypercapnic environment and examined the responses of anabolic genes and proteolytic systems. Hypercapnia caused decreases in muscle fiber diameter, protein content and force production, which indicated muscle atrophy. Similarly, the increased level of CO2 caused thinner myotubes, lower protein content and decreased anabolic signaling such as rDNA transcription rate. Meanwhile, the activity of ubiquitin-proteasome system was increased due to the increased AMPK-FOXO3- MuRF1 pathway. These results suggest that the coordination of protein synthesis and degradation is of great importance in the dynamic process of protein modification. To further understand the regulation of muscle hypertrophy at the molecular level, we generated conditional skeletal muscle specific c-Myc knock out mice. In Paper III, we found that the mice lacking c-Myc displayed normal post-natal skeletal muscle development in terms of body weight, muscle weight, RNA content and rDNA transcription rate when compared to control mice. To challenge the c-Myc knock out mice in a rapid growth situation, we performed synergist ablation of the gastrocnemius and soleus muscles to induce compensatory hypertrophy of the plantar flexor muscles. The c-Myc conditional knock out mice showed normal hypertrophic response, which indicated that c-Myc is dispensable for hypertrophic growth in terminally differentiated cells. We further confirmed this finding by blocking c-Myc function via chemical inhibitors in C2C12 myotubes. However, inhibiting c-Myc by siRNA or loss of its function by chemical inhibitor in proliferating C2C12 myoblast suggested a different role by blocking cell proliferation and decrease rDNA transcription. Therefore, we suggest a cell stage-specific role of c-Myc in the regulation of growth/proliferation and rDNA transcription in the myogenic cell lines. To further understand the regulation of ribosome biogenesis during muscle hypertrophy, we focused on the mTOR pathway due to its regulatory role in protein homeostasis. In Paper IV, the mature C2C12 myotubes were stimulated with high concentration of serum to induce myotube hypertrophy. Increased mTOR signaling was detected during hypertrophy and the inhibition of mTOR prevented the hypertrophic response. When inhibiting the function of mTOR, rDNA transcription rate was decreased as well as the mTOR/rDNA promoter binding, which indicated a role for nuclear mTOR in the regulation of rDNA transcription. Furthermore, chemical inhibition of RNA Polymerases I prevented rRNA accumulation and myotubes hypertrophy, suggesting that intact Pol I mediated transcription was necessary for muscle hypertrophy. In conclusion, this thesis investigates the regulation of skeletal muscle hypertrophy by different mechanisms. It highlights the importance of ribosome biogenesis during skeletal muscle hypertrophy.