Closed projects

Circular RNAs (circRNAs) are abundantly expressed in physiology and disease remains largely unknown. Recent studies and our own work report an increasing number of circRNAs with potential functions in the cardiovascular (CV) system. However, little is known about the protein factors involved in their biogenesis and their molecular mode of action. The aim of this project is to identify and characterise the hypoxia-controlled circRNA biogenesis and functions in endothelial cells (ECs). In the first part of the application, we will study the role of the previously identified circRNA cZNF292, which is induced by hypoxia and regulates angiogenesis in vitro. Specifically, we will characterise the mechanism of action by bioinformatics analysis and biochemical assays to investigate potential cZNF292-interacting proteins. In vivo functions will be determined by generation of knock-out mice, which specifically lack the circular RNA but express the linear transcript. In the second part, we will globally evaluate the regulation of circRNA splicing in endothelial cells. To this end, we will detect endothelial-enriched circRNAs, which show a high circRNA/host gene ratio, from RNA-Seq data and use machine learning to extract critical RNA sequence and structure features of regulated circRNAs. In order to identify putative regulatory RNA binding proteins (RBPs), in silico binding site predictions will be integrated with public CLIP-seq datasets (ENCODE Project) and RBP expression profiles in the CV system. Promising candidates will then be investigated experimentally.

CircRNAs are a novel class of long non-coding RNAs, originating from splicing of a downstream 5’ to an upstream 3’ splice site, leading to covalently closed RNA loops. Previous work has characterised several human circRNAs, which are differentially expressed in cardiovascular (CV) cells, and which might be linked to CV health and disease. To date, little is known about the regulation of circRNAs during CV cell development and how they might control disease-related cell functions. In our previous work, we have delineated the molecular mechanism of circular isoforms of the long non- coding RNA ANRIL (circANRIL) in atherosclerosis. We also found that circANRIL becomes upregulated during differentiation of iPSCs and promotes atheroprotective cell functions in mature SMCs and macrophages. In preliminary work to this proposal, we have established protocols to differentiate iPSC into various cell types relevant in the CV system, such as endothelial cells (ECs) and SMCs. As proof of concept, we found several hundred differentially expressed circRNAs by ultra-deep next generation sequencing (NGS) in RNA collected during iPSC differentiation to SMC. To study their functional role in cell development and in mature CV cells, we aim to (1) systematically identify relevant circRNAs and their regulators using NGS and proteome analyses in ECs and SMCs, (2) modulate their expression using established overexpression and knockdown approaches and test their effect on iPSC differentiation and CV cell function, and (3) translate these findings to the in vivo situation. The proposed project will improve our understanding of the role of circRNAs in CV health and disease.

During embryogenesis, different cell types assemble to give rise to a functional heart. While the role of many protein-coding RNAs in controlling the gene regulatory networks governing heart development is well established, the class of long non-coding RNAs (lncRNAs) and their role in heart development and disease has only recently shifted into focus. Here we aim at genetically analyzing and determining the mechanisms of so far undescribed lncRNA loci in heart development and heart function in vivo. Our work can uncover novel mechanisms how lncRNA loci are participating in controlling gene networks during development of a complex organ such as the heart. We generated a distinct expression profile of lncRNAs, differentially expressed in pluripotent mouse embryonic stem cells, early Mesp1 positive cardiac progenitor cells and embryonic (E) 8.5/E9.0 heart tube. From this dataset, we selected a so far undescribed lncRNA locus specifically expressed from early heart tube stages onwards. We termed this locus Sweetheart (Swht). Upon generation of a transcriptional Swht mutant mouse, we found that the Swht gene is dispensable for embryonic development, but plays an important role in recovery after myocardial injury. We could establish that its function depends on the RNA transcript, as transgenic re-expression of the Swht RNA in mice rescues this effect. This enables us now to identify the active part of the RNA and establish the underlying paradigm of its function.

Hypoxia inducible factors (HIFs) are heterodimeric transcription factors composed of HIFα and HIF1β subunits that occupy central roles in regulating oxygen homeostasis and the pathogenesis of human disease including cancer and cardiovascular (CV) disease. They are activated in hypoxic tissue to induce a transcriptional program embracing coding and non-coding RNA transcripts that are entrusted to modulate both the supply and consumption of oxygen. To date, it is unclear if HIFs also activate the transcription of enhancer RNAs (eRNAs) to afford cell- and signal-specificity of selected HIF output responses. In the present proposal, we aim to study the physiologic impact and mechanistic underpinnings of cardiac-specific eRNAs identified in our genome-wide screen for HIF-dependent functional eRNAs in heart disease. In preliminary data, we validated the first candidate – hypoxia-inducible factor (HIF)1α-activated eRNA (HERNA1) that is robustly induced in pathologic stress-induced mouse models of human cardiac hypertrophy. In vivo administration of antisense oligonucleotides (ASO) targeting Herna1 protected mice from stress-induced pathologic cardiac hypertrophy, while their delivery post-disease development reversed left ventricular growth and dysfunction, resulting in increased overall survival. Based on these findings, we aim to embark on a research program to assess the pathophysiologic relevance of 2 further eRNAs that were identified by the screen. The impact of these eRNAs on cardiac growth, metabolism and function will be assessed in human iPS-derived cardiac organoids by silencing with ASO and CRISPR/Cas-mediated eRNA inactivation. Subsequently, deep functional characterisation of these eRNAs on cardiac pathogenesis and progression will be determined by ASO treatment and gene deletion by CRISPR/Cas in mouse surgery-induced models of aortic stenosis- and hypertension-induced cardiac hypertrophy. This project aims to identify and target clinically relevant eRNAs for the treatment of heart disease.

Ventricular arrhythmias are a common cause of sudden cardiac death, which accounts for 50% of all cardiac deaths. In contrast to other cardiovascular disease entities, the role of ncRNAs in cardiac arrhythmia is largely unclear. We have identified miR-365 as a microRNA that targets repolarizing ion channels in cardiac myocytes and is abundant in these cells in humans. In addition, we established a disease model with human genetic background, using patient-specific induced pluripotent stem cells that are differentiated to cardiac myocytes (iPSC-CMs). In this project, we will functionally characterise miR-365 and further ncRNAs that we have meanwhile identified. For miR-365, we will apply synthetic mimics or inhibitors, next to CRISPR-based genetic inactivation, and determine alterations in the action potential. As a reporter for action potential duration (APD), voltage-sensitive fluorescent protein (VSFP)-based FRET imaging is employed, which allows for the monitoring of individual cells in large numbers. In addition, patch-clamp analyses in smaller cell numbers will resolve action potential (AP) characteristics in more detail. We will also study the regulation of miR-365 in human failing myocardium, and test in iPSC-CMs from patients with QT syndromes whether manipulation of miR-365 alleviates the arrhythmic phenotype. Further, we will evaluate the role if miR-365 in electric remodelling of cardiac myocytes, since we found that pathologic elevation of miR-365 markedly increased the occurrence of disturbed electric automaticity. In addition, our preliminary bioinformatic analyses on predicted ncRNA-target regulatory networks as well as bulk and single cell transcriptome analysis on iPSC-CMs pointed to new ncRNA candidates associated with cardiac myocyte electrophysiology. We plan to validate the potential role of these novel ncRNAs by identifying their bona fide targets, before applying CRISPR inactivation in VSFP- sensor iPSC-CMs for functional analysis. This project will, in unprecedented depth, resolve the function of ncRNAs in cardiac repolarization and provide a basis for the development of ncRNA-based therapies.