Dual Translational Control in Cardiomyocytes by Heterogeneous mTORC1 and Hypertrophic ERK Activation

Background

Cardiac hypertrophy allows post-mitotic cardiomyocytes to meet increased hemodynamic demands but can predispose the heart to adverse clinical outcomes. Despite its central role in cardiac adaptation, the translational control mechanisms that drive cardiac hypertrophy are poorly understood. In this study, we elucidate the relative contributions of the various translational control mechanisms operant during homeostasis and hypertrophic growth.

Methods

A combination of immunofluorescence and single myocyte protein synthesis assays were used to dissect the single-cardiomyocyte mechanisms of translational control under basal and hypertrophic conditions in isolated adult rat cardiomyocytes. Translational control mechanism were examined in a mouse model of acute hypertrophic phenylephrine (PE) stimulation prior to overt cardiac growth.

Results

We observed strikingly heterogeneous activity of mTORC1, the master regulator of translation, across cardiomyocytes both in situ and ex vivo . Heterogeneous mTORC1 activity drove heterogeneous protein synthesis, with translation primarily controlled via canonical mTORC1-dependent 4EBP1 phosphorylation at Thr36/Thr45/Thr69 under baseline conditions. Hypertrophic PE stimulation recruited more cardiomyocytes into a high mTORC1 activity state. PE induced a switch in 4EBP1 phosphorylation by increasing mTORC1-dependent phosphorylation at Thr36/Thr45, but not Thr69. Further, PE induced a novel mTORC1-independent, but MEK-ERK-dependent, pathway driving 4EBP1 phosphorylation at Ser64 in both isolated cardiomyocytes and in vivo . Ribosome biogenesis was also observed within hours upon hypertrophic stimulation, while the mTORC1-S6K-eEF2K-eEF2 pathway was not found to be a major driver of protein translation.

Conclusions

Protein synthesis is heterogeneous across cardiomyocytes driven by heterogeneous mTORC1 activity. MEK-ERK signaling directly controls 4EBP1 phosphorylation to augment translation during cardiac hypertrophy and challenges the canonical model of translation initiation.

Key points:

• mTORC1 activity is low and heterogeneous across cardiomyocytes at baseline.

• Heterogeneous mTORC1 activity drives variable 4EBP1 phosphorylation at Thr36/Thr45/Thr69, resulting in heterogeneous protein translation.

• Phenylephrine stimulation recruits more cardiomyocytes into a high mTORC1 activity state to augment protein synthesis through 4EBP1 phosphorylation at Thr36/Thr45, but not Thr69.

• Phenylephrine stimulation boosts protein translation through mTORC1-independent but ERK-dependent phosphorylation of 4EBP1 at Ser64.

• mTORC1-S6K-eEF2K-eEF2 pathway is not a major driver of global protein translation.

• Ribosome biogenesis is observed within hours after hypertrophic stimulation.

• The data demonstrates a dual input model of mTORC1 and ERK dependent phosphorylation of 4EBP1 to regulate protein translation in cardiomyocytes.