Post-transcriptional and translational mechanisms of cardiac growth

1. Identification of dynamic RNA-binding proteins in primary cardiomyocytes uncovers Cpeb4 as a regulator of cardiac growth Mutations or decreased expression of mRNA-binding proteins (mRBPs) can lead to cardiomyopathies in humans. The present study defined the first compendium of dynamically binding mRBPs in healthy versus diseased primary cardiomyocytes at a system-wide level by RNA interactome capture. Among these mRBPs, Cytoplasmic polyadenylation element binding protein 4 (Cpeb4) was defined as a dynamic mRBP in diseased cardiomyocytes, and was found to regulate cardiac growth both in vitro and in vivo. To investigate the functions of Cpeb4 in cardiomyocytes, mRNAs bound to and regulated by Cpeb4 were identified. These data implicate that Cpeb4 regulates transcriptional activity by differential translation of transcription factors involved in cellular remodeling in response to pathological growth stimulation. Among Cpeb4 target RNAs, two Zinc finger transcription factors (Zeb1 and Zbtb20) were identified. The present study shows that Cpeb4 regulates the translation of these mRNAs and that Cpeb4 depletion increases their expression. Thus, Cpeb4 emerges as critical regulator of myocyte function by differential binding of specific mRNAs in response to pathological growth stimulation. 2. mTOR-proteasomal dysfunction following deletion of Pras40 inhibits cardiac growth but results in cardiac failure The mammalian target of Rapamycin complex 1 (mTORC1) increases cell size by initiating translation as well as by inhibiting catabolic functions such as proteolysis and autophagy. A previous study from our lab proposed Proline-rich Akt substrate 1 (Pras40) as a cardioprotective, endogenous inhibitor of mTOR-dependent protein synthesis during pathological growth. Pras40 is released from mTORC1 during growth, but other interactions are largely unknown. The present study aims at understanding the molecular mechanism of Pras40 to cardiac growth and function. To test consequences of Pras40 deletion on cardiac function in vivo, two novel Pras40 knock-out mice were subjected to pathological and physiological hypertrophy (Transverse aortic constriction, swimming). Conversely to Pras40 overexpression, growth was significantly blunted in KO animals and function reduced. mTORC1 signaling as well as proteasomal function were severely disturbed in KO animals. Mechanistically, chymotrypsin-like 26S proteasomal activity was blunted in KO hearts as well as isolated cardiomyocytes from KO animals. Disturbed proteasomal function in KO mice lead to severe alterations in metabolic functions highlighting the importance of both intact mTORC1 signaling and proper proteasomal maintenance during cardiac stress. Reactivation of proteasomal activity in vivo in KO mice restored cardiac function to WT levels, and overexpression of mutant, mTOR-released Pras40 had a similar effect. The present study provides evidence that Pras40 links anabolic protein synthesis and catabolic proteolysis in the heart: At rest, Pras40 binds and inhibits mTOR, but when released during pathological growth, Pras40 directly interacts with the 26S proteasome and modulates its activity