PRAS40 suppresses atherogenesis through inhibition of mTORC1-dependent pro-inflammatory signaling in endothelial cells

Endothelial pro-inflammatory activation plays a pivotal role in atherosclerosis, and many pro-inflammatory and atherogenic signals converge upon mechanistic target of rapamycin (mTOR). Inhibitors of mTOR complex 1 (mTORC1) reduced atherosclerosis in preclinical studies, but side effects including insulin resistance and dyslipidemia limit their clinical use in this context. Therefore, we investigated PRAS40, a cell type-specific endogenous modulator of mTORC1, as alternative target. Indeed, we previously found PRAS40 gene therapy to improve metabolic profile; however, its function in endothelial cells and its role in atherosclerosis remain unknown. Here we show that PRAS40 negatively regulates endothelial mTORC1 and pro-inflammatory signaling. Knockdown of PRAS40 in endothelial cells promoted TNFα-induced mTORC1 signaling, proliferation, upregulation of inflammatory markers and monocyte recruitment. In contrast, PRAS40-overexpression blocked mTORC1 and all measures of pro-inflammatory signaling. These effects were mimicked by pharmacological mTORC1-inhibition with torin1. In an in vivo model of atherogenic remodeling, mice with induced endothelium-specific PRAS40 deficiency showed enhanced endothelial pro-inflammatory activation as well as increased neointimal hyperplasia and atherosclerotic lesion formation. These data indicate that PRAS40 suppresses atherosclerosis via inhibition of endothelial mTORC1-mediated pro-inflammatory signaling. In conjunction with its favourable effects on metabolic homeostasis, this renders PRAS40 a potential target for the treatment of atherosclerosis

Genomic structural variations lead to dysregulation of important coding and non-coding RNA species in dilated cardiomyopathy

The transcriptome needs to be tightly regulated by mechanisms that include transcription factors, enhancers, and repressors as well as non-coding RNAs. Besides this dynamic regulation, a large part of phenotypic variability of eukaryotes is expressed through changes in gene transcription caused by genetic variation. In this study, we evaluate genome-wide structural genomic variants (SVs) and their association with gene expression in the human heart. We detected 3,898 individual SVs affecting all classes of gene transcripts (e.g., mRNA, miRNA, lncRNA) and regulatory genomic regions (e.g., enhancer or TFBS). In a cohort of patients (n = 50) with dilated cardiomyopathy (DCM), 80,635 non-protein-coding elements of the genome are deleted or duplicated by SVs, containing 3,758 long non-coding RNAs and 1,756 protein-coding transcripts. 65.3% of the SV-eQTLs do not harbor a significant SNV-eQTL, and for the regions with both classes of association, we find similar effect sizes. In case of deleted protein-coding exons, we find downregulation of the associated transcripts, duplication events, however, do not show significant changes over all events. In summary, we are first to describe the genomic variability associated with SVs in heart failure due to DCM and dissect their impact on the transcriptome. Overall, SVs explain up to 7.5% of the variation of cardiac gene expression, underlining the importance to study human myocardial gene expression in the context of the individual genome. This has immediate implications for studies on basic mechanisms of cardiac maladaptation, biomarkers, and (gene) therapeutic studies alike

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

m(6)A-mRNA methylation regulates cardiac gene expression and cellular growth

Conceptually similar to modifications of DNA, mRNAs undergo chemical modifications, which can affect their activity, localization, and stability. The most prevalent internal modification in mRNA is the methylation of adenosine at the N-6-position (m(6)A). This returns mRNA to a role as a central hub of information within the cell, serving as an information carrier, modifier, and attenuator for many biological processes. Still, the precise role of internal mRNA modifications such as m(6)A in human and murine-dilated cardiac tissue remains unknown. Transcriptome-wide mapping of m(6)A in mRNA allowed us to catalog m(6)A targets in human and murine hearts. Increased m(6)A methylation was found in human cardiomyopathy. Knockdown and overexpression of the m(6)A writer enzyme Mettl3 affected cell size and cellular remodeling both in vitro and in vivo. Our data suggest that mRNA methylation is highly dynamic in cardiomyocytes undergoing stress and that changes in the mRNA methylome regulate translational efficiency by affecting transcript stability. Once elucidated, manipulations of methylation of specific m(6)A sites could be a powerful approach to prevent worsening of cardiac function

Genomic structural variations lead to dysregulation of important coding and non‐coding RNA species in dilated cardiomyopathy

Abstract The transcriptome needs to be tightly regulated by mechanisms that include transcription factors, enhancers, and repressors as well as non‐coding RNAs. Besides this dynamic regulation, a large part of phenotypic variability of eukaryotes is expressed through changes in gene transcription caused by genetic variation. In this study, we evaluate genome‐wide structural genomic variants (SVs) and their association with gene expression in the human heart. We detected 3,898 individual SVs affecting all classes of gene transcripts (e.g., mRNA, miRNA, lncRNA) and regulatory genomic regions (e.g., enhancer or TFBS). In a cohort of patients (n = 50) with dilated cardiomyopathy (DCM), 80,635 non‐protein‐coding elements of the genome are deleted or duplicated by SVs, containing 3,758 long non‐coding RNAs and 1,756 protein‐coding transcripts. 65.3% of the SV‐eQTLs do not harbor a significant SNV‐eQTL, and for the regions with both classes of association, we find similar effect sizes. In case of deleted protein‐coding exons, we find downregulation of the associated transcripts, duplication events, however, do not show significant changes over all events. In summary, we are first to describe the genomic variability associated with SVs in heart failure due to DCM and dissect their impact on the transcriptome. Overall, SVs explain up to 7.5% of the variation of cardiac gene expression, underlining the importance to study human myocardial gene expression in the context of the individual genome. This has immediate implications for studies on basic mechanisms of cardiac maladaptation, biomarkers, and (gene) therapeutic studies alike

TIP30 counteracts cardiac hypertrophy and failure by inhibiting translational elongation

Abstract Pathological cardiac overload induces myocardial protein synthesis and hypertrophy, which predisposes to heart failure. To inhibit hypertrophy therapeutically, the identification of negative regulators of cardiomyocyte protein synthesis is needed. Here, we identified the tumor suppressor protein TIP30 as novel inhibitor of cardiac hypertrophy and dysfunction. Reduced TIP30 levels in mice entailed exaggerated cardiac growth during experimental pressure overload, which was associated with cardiomyocyte cellular hypertrophy, increased myocardial protein synthesis, reduced capillary density, and left ventricular dysfunction. Pharmacological inhibition of protein synthesis improved these defects. Our results are relevant for human disease, since we found diminished cardiac TIP30 levels in samples from patients suffering from end‐stage heart failure or hypertrophic cardiomyopathy. Importantly, therapeutic overexpression of TIP30 in mouse hearts inhibited cardiac hypertrophy and improved left ventricular function during pressure overload and in cardiomyopathic mdx mice. Mechanistically, we identified a previously unknown anti‐hypertrophic mechanism, whereby TIP30 binds the eukaryotic elongation factor 1A (eEF1A) to prevent the interaction with its essential co‐factor eEF1B2 and translational elongation. Therefore, TIP30 could be a therapeutic target to counteract cardiac hypertrophy