Introduction:
Translation as well as the metabolic activity within the cell are fundamental processes which are highly regulated. At the interface of those two processes lies the kinase complex mTORC1 sensing nutrient availability to regulate translation. It has several downstream effectors including the eukaryotic initiation factor 4G1 (eIF4G1) which is known to serve as a central scaffold for several proteins during translation initiation. Its regulation occurs through phosphorylation at Ser1147, Ser1187 and Ser1231. mTORC1 inhibition was previously shown to attenuate cardiac hypertrophy and cardiac dysfunction. However, the occurrence of side effects demands for alternative approaches such as targeting downstream effectors.
Methods:
Protein synthesis as well as metabolic activity was studied after eIF4G1 knockdown by siRNA. Translation was assessed through a puromycin incorporation assay by treating cells with puromycin 30 min before lysis. The metabolic activity over time was evaluated by an MTT assay administrating MTT at different timepoints (0h, 24h, 96h and 120h). To imitate nutrient availability versus a starvation state, cells were either serum starved or cultured in serum supplemented with 10% fetal calf serum (FCS). Experiments were analysed in a time dependent manner starving the cells first and then changing the media to 10% FCS at different time points. Dependence of the response to serum on mTORC1 was tested by adding rapamycin, an mTORC1 inhibitor, to the cells. Hypertrophic growth of cardiomyocytes was induced with phenylephrine (PE).
Results:
mTORC1 dependent translation under growth conditions was analysed within Hela and Hek cell lines and primary cardiomyocytes. In HEK/Hela cells, experiments showed that on the one hand knockdown of eIF4G1 did not show any impact on bulk protein synthesis but on the other hand led to a decreased metabolic activity within the cells over time. First experiments testing the dependence of metabolic activity on mTORC1-dependet regulation of eIF4G1 showed that eIF4G1 phosphorylation was reduced upon rapamycin administration and in a starvation state of the cell. Serum starvation was also tested in cardiomyocytes which showed similar results. Exposing cardiomyocytes to nutrients for varying time spans revealed that prolonged serum stimulation increased mTORC1-mediated eIF4G1 phosphorylation. Although Hek/Hela cells showed no difference of baseline protein synthesis rates after eIF4G1 knockdown, cardiomyocytes that underwent hypertrophic growth after PE administration showed reduced protein synthesis after knockdown of eIF4G1, indicating that eIF4G1 is essential for increased translation rates during cardiomyocyte hypertrophy.
Conclusion:
We here show that eIF4G1 plays a central role in cellular metabolism and protein synthesis during cardiomyocyte growth. We hypothesize that the activity of eIF4G1 is directly regulated through mTORC1-dependent phosphorylation and will further explore whether this pathway can be targeted to attenuate pathological hypertrophic remodelling during heart disease.
