Abstract 174: Ras Homologue Enrich in Brain (Rheb) Enhances Hypertrophic Response via the Mammalian Target of Rapamycin (mTOR)/p70S6K Pathway
Rheb (Ras homologue enrich in brain) is a small GTP binding protein, which is abundantly expressed in the heart. Although Rheb is active in unstimulated cells, its activity is negatively regulated by tuberous sclerosis complex 2. Rheb stimulates mammalian target of rapamycin (mTOR), a master regulator of protein translation, thereby potentially regulating protein synthesis in cardiac myocytes. The goal of this study was to elucidate the involvement of Rheb in cardiac hypertrophy. Treatment with phenylephrine (PE), a hypertrophic agonist, for 48 h significantly increased protein levels of Rheb in cultured neonatal cardiac myocytes (3.5 fold, p<0.05). In order to clarify the role of Rheb upregulation, we transduced adenovirus harboring Rheb (Ad-Rheb) or LacZ (Ad-LacZ) into cultured cardiac myocytes. Ad-Rheb significantly increased Thr 398 phosphorylation of p70 S6K, indicating that the Rheb-mTOR pathway is activated (2.8±0.2 fold vs Ad-LacZ, p<0.05). Ad-Rheb significantly increased the myocyte size and the protein content (1.7±0.2 and 1.8±0.1 fold, p<0.05), which was comparable to stimulation with PE (1.9±0.3 and 1.9±0.1 fold). Ad-Rheb also enhanced the activity of the ANF-luciferase reporter (3.8±0.9 fold, p<0.05), which was not as strong as PE treatment (8.2±2.1 fold, p<0.05). Similarly, although Ad-Rheb enhanced sarcomeric organization of cardiac myocytes, it was not as strong as PE. Adenovirus harboring Rheb S20N (Ad-R20N) reduced Thr 398 phosphorylation of p70 S6K in the presence of serum, suggesting that Rheb S20N acts as a dominant negative. Transduction of Ad-S20N markedly reduced PE-induced increases in cell size and protein content (1.2±0.1 and 1.1±0.1 fold). Ad-S20N partially but significantly suppressed PE-induced increases in ANF-luciferase activity (2.5±0.3 fold, p<0.05 vs PE alone 8.2±2.1 fold) and sarcomeric organization. mRNA level of Rheb was also enhanced in the mouse heart subjected to thoracic aortic banding (30.3±10.8 fold, p<0.05). These results suggest that Rheb is upregulated by hypertrophic stimuli and stimulates the mTOR-p70S6K pathway in cardiac myocytes. Furthermore, Rheb plays an essential role in mediating cardiac hypertrophy by primarily regulating the cell size and protein content.