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Exploring selected long non-coding RNAs as diagnostics and therapeutic targets for heart failure

Periodic Reporting for period 3 - LONGHEART (Exploring selected long non-coding RNAs as diagnostics and therapeutic targets for heart failure)

Reporting period: 2018-06-01 to 2019-11-30

Cardiac stress such as myocardial infarction or hypertension leads to cellular “remodeling” of the left ventricle resulting in heart failure. Protein-coding genes originate from only 1.5% of the genome, whereas the larger remaining portion is often transcribed to non-coding RNAs, of which functional importance is still ill understood. We pioneered a role of small microRNAs as diagnostics and therapeutic targets for heart failure (Nature, 2008; Nature Comm, 2012, J Clin Invest, 2014). We now will focus on the larger fraction of long non-coding RNAs (lncRNAs) and their functional roles, as well as diagnostic and therapeutic use in heart failure.
Despite clinical advances, diseases of the cardiovascular system are the most common cause of morbidity and mortality in the EU with currently up to 50 million people suffering from heart failure. These important challenges call for a better understanding of underlying mechanisms to enable development of innovative, effective diagnostic and therapeutic strategies for heart failure.
The proposal has the following interconnected objectives: a) identify novel functional relevant cardiac remodeling-associated lncRNAs; b) characterise key lncRNA cardiac targetomes; c) investigate lncRNA-paracrine mechanisms and the diagnostic and prognostic potential of cardiac-derived extracellular lncRNAs using large clinical cohorts; and d) discover their therapeutic potential to prevent cardiac remodeling in clinically relevant animal models. Innovative molecular and cell-based methods, a unique lncRNA-shRNA library, genetic animal models and availability of large clinical biobanks will form the basis for a successful strategy. LONGHEART will lead to ground-breaking new insight into the role of lncRNAs in the heart. These findings will firmly establish lncRNA-based mechanisms to identify fundamentally novel diagnostic and therapeutic entry points for a most serious clinical important disorder in dire need for new diagnostic and therapeutic paradigms.
Within LONGHEART we are within our timelines, personal has been successfully recruited and we do not see any problems for delays or other problems.
We have started to focus on the large fraction of long non-coding RNAs (lncRNAs) and their functional roles, as well as diagnostic and therapeutic use in heart failure. The proposal has the following interconnected objectives and I will briefly describe the current status of our work
a) Identify novel functional relevant cardiac remodeling-associated lncRNAs;
Status: We have implemented novel high-throughput methods for lncRNA identification and already identified and patented several lncRNA candidates as interesting novel therapeutic targets. For instance, by using global lncRNA expression profiling we found several lncRNA transcripts to be deregulated during pressure overload-induced cardiac hypertrophy in mice. Using stringent selection criteria, we identified Chast (cardiac hypertrophy-associated transcript) as a potential lncRNA candidate that influences cardiomyocyte hypertrophy. Cell fractionation experiments indicated that Chast is specifically up-regulated in cardiomyocytes in vivo in transverse aortic constriction (TAC)-operated mice. In accordance, CHAST homolog in humans was significantly up-regulated in hypertrophic heart tissue from aortic stenosis patients and in human embryonic stem cell-derived cardiomyocytes upon hypertrophic stimuli. Viral-based overexpression of Chast was sufficient to induce cardiomyocyte hypertrophy in vitro and in vivo. GapmeR-mediated silencing of Chast both prevented and attenuated TAC-induced pathological cardiac remodeling with no early signs on toxicological side effects. Mechanistically, Chast negatively regulated Pleckstrin homology domain-containing protein family M member 1 (opposite strand of Chast), impeding cardiomyocyte autophagy and driving hypertrophy. These results indicate that Chast can be a potential target to prevent cardiac remodeling and highlight a general role of lncRNAs in heart diseases. This study has been recently published in the prestigious journal Science Translational Medicine (Viereck et al., Science Transl Med. 2016 Feb 17;8(326):326ra22. doi: 10.1126/scitranslmed.aaf1475). In addition, a set of further lncRNAs have been identified that are currently under investigation within LONGHEART. One other example is our recent screen of lncRNAs in cardiac fibroblasts, another frequent cell type within the heart. Indeed, cardiac fibroblasts (CFs) drive extracellular matrix remodeling after pressure overload, leading to fibrosis and diastolic dysfunction. We characterized lncRNA expression in murine CFs after chronic pressure overload to identify CF-enriched lncRNAs and investigate their function and contribution to cardiac fibrosis and diastolic dysfunction. This global lncRNA profiling identified several dysregulated transcripts. Among them, the lncRNA maternally expressed gene 3 (Meg3) was found to be mostly expressed by CFs and to undergo transcriptional downregulation during late cardiac remodeling. In vitro, Meg3 regulated the production of matrix metalloproteinase-2 (MMP-2). GapmeR-mediated silencing of Meg3 in CFs resulted in the downregulation of Mmp-2 transcription, which, in turn, was dependent on P53 activity both in the absence and in the presence of transforming growth factor-β I. Chromatin immunoprecipitation showed that further induction of Mmp-2 expression by transforming growth factor-β I was blocked by Meg3 silencing through the inhibition of P53 binding on the Mmp-2 promoter. Consistently, inhibition of Meg3 in vivo after transverse aortic constriction prevented cardiac MMP-2 induction, leading to decreased cardiac fibrosis and improved diastolic performance. Collectively, our findings uncover a critical role for Meg3 in the regulation of MMP-2 production by CFs in vitro and in vivo, identifying a new player in the development of cardiac fibrosis and potential new target for the prevention of cardiac remodeling. These findings also have been patented to secure possibility for future clinical developments. Data have been published in Circ Res. 2017 Aug 18;121(5):575-583.
To search for functional lncRNAs in a more global manner we did the following approach; we created a lentiviral shRNA library-based approach for functional lncRNA profiling. We validated our library approach in NIH3T3 (3T3) fibroblasts by identifying lncRNAs critically involved in cell proliferation. Using stringent selection criteria we identified lncRNA NR_015491.1 out of 3842 different RNA targets represented in our library. We termed this transcript Ntep (non-coding transcript essential for proliferation), as a bona fide lncRNA essential for cell cycle progression. Inhibition of Ntep in 3T3 and primary fibroblasts prevented normal cell growth and expression of key fibroblast markers. Mechanistically, we discovered that Ntep is important to activate P53 concomitant with increased apoptosis and cell cycle blockade in late G2/M. Our findings suggest Ntep to serve as an important regulator of fibroblast proliferation and function. In summary, our study demonstrates the applicability of an innovative shRNA library approach to identify long non-coding RNA functions in a massive parallel approach. Results of this study have been published recently (Cell Death Differ. 2018 Feb;25(2):307-318.).
In addition we now have started to search also for circular RNAs, which are long noncoding RNAs forming circles de to backsplicing events. A first paper on several circRNAs involved in cardiac atrophy has been published (Circ Res. 2017 Nov 13. pii: CIRCRESAHA.117.311335. doi: 10.1161/CIRCRESAHA.117.311335).

b) Characterize key lncRNA cardiac targetomes;
Status: We have started to use lncRNA-pulldowns to study lncRNA/protein interactions. First data have been already published (Viereck et al., Science Transl Med. 2016 Feb 17;8(326):326ra22. doi: 10.1126/scitranslmed.aaf1475). Specifically, we synthesized biotinylated lncRNA transcripts, linear PCR fragments were prepared with the Expand High Fidelity PCR System. Biotin-labled RNA of Chast and the control transcripts [luciferase (luc) or an artificial sequence complementary to Chast (Chast-as)] were generated using the Ampliscribe T7-Flash Biotin-RNA transcription Kit according to the manufacturer’s specification. Biotinylated RNAs were treated with RNAse-free DNAse I and purified by Ammonium acetate precipitation. Quality of the transcripts was analyzed by non-denaturing gel electrophoresis and SYBR Green II RNA Gel Stain. The RNA-pulldown assays using biotinylated Chast constructs indicated that Chast interacts with several cardiac-relevant proteins. Interestingly, pathway enrichment analysis of the interacting proteins using Enrichr revealed that Chast interacts mainly with proteins involved in cardiomyopathies. These bioinformatic and experimental screening tools revealed a strong correlation (as quantified by main bioinformatics hits of cardiac disease pathways rather than of others) of the lncRNA Chast with cardiac disease pathways, qualifying Chast as a potential target for therapeutics. A number of other studies examining the interaction of proteins with various lncRNAs are currently underway.

c) Investigate lncRNA-paracrine mechanisms and the diagnostic and prognostic potential of cardiac-derived extracellular lncRNAs using large clinical cohorts;
Status: We have started to discover lncRNAs secreted by hypoxic cardiomyocytes and first results have been presented in a Keystone meeting (March 2017), which I was organizing (Keystone, Colorado. USA). Specifically, we have unraveled a novel intercellular communication route between hypoxic cardiomyocytes and fibroblasts via extracellular vesicles, which are enriched with lncRNAs. In particular, we have identified two hypoxia-responsive lncRNAs: lncRNA_E16 was shown to be predominantly transported to fibroblasts via exosomal transfer, whereas lncRNA Neat1 was rather enriched in microvesicles during hypoxic conditions, indicating a selective sorting process of different lncRNAs. In vivo data of extracellular vesicles derived from mouse hearts after myocardial infarction confirmed the selective packaging mechanism. In addition, we investigated their impact on fibroblast function and could show that both lncRNAs are involved in the fibrotic response. Furthermore, we identified that the extracellular transfer of Neat1 provided a new intercellular signalling mechanism in order to contribute to fibroblast survival and to regulate cell-cycle progression. In addition, we investigated the regulatory upstream mechanisms of Neat1 expression and could show that Neat1 is a downstream target of HIF2A under hypoxic conditions. Moreover, we identified Neat1 as a P53 bona fide target under basal conditions. Correlation of our in vitro data to patient data of cardiac disease provided first insights into potential clinical applications. We identified circulating 7sk, the human homologue of lncRNA_E16, as a potential prognostic marker in plasma samples of patients with and without left ventricular remodelling. In addition, human Neat1 expression was also increased in myocardial tissue of heart failure patients, suggesting a clinical relevance for this lncRNA. Apart from the investigation of lncRNA function, co-culture assays were performed to study the EV-mediated crosstalk in more detail. We showed that conditioned medium of hypoxic cardiomyocytes triggered the pro-fibrotic response in fibroblasts, while depletion of vesicles out of the medium attenuated this effect. Collectively, we could show that in response to hypoxia, cardiomyocytes secrete and release different types of extracellular vesicles, which are enriched in lncRNAs and are taken up by fibroblasts, affecting fibroblast biology and might serve as promising therapeutic tools in the future. These findings will help to decipher the molecular mechanisms governing the functional consequences of EVs in the infarcted heart in order to design new powerful therapeutic tools for treating cardiac ischemic diseases.
d) Discover their therapeutic potential to prevent cardiac remodeling in clinically relevant animal models.
Status: We have developed AAV-based cell type specific targeting vectors for the modulation of cardiac lncRNAs and have provided first evidence for therapeutic manipulation of lncRNAs in heart diseases (Viereck et al., Science Transl Med. 2016 Feb 17;8(326):326ra22. doi: 10.1126/scitranslmed.aaf1475). In addition we have designed ad validated several lncRNA inhibiting compounds such as so called GAPMERs. Specifically, we could show that in vivo treatment of mice after pressure overload of the left ventricle develop significantly less cardiac fibrosis and have improved diastolic function. Data have been published recently (Circ Res. 2017 Nov 13. pii: CIRCRESAHA.117.311335. doi: 10.1161/CIRCRESAHA.117.311335). Using global transcriptome profiling from murine myocardium exposed to doxorubicin identified five differentially expressed RNA binding proteins. We became interested in RNA binding proteins because of their described rile in the regulation of circRNA expression. Indeed, expression of the RNA-binding protein Quaking (QKI) in response to doxorubicin was strongly downregulated in rodent cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes in vitro and in vivo in mice. AAV9-mediated cardiac overexpression of Qki5 prevented cardiac apoptosis and cardiac atrophy induced by doxorubicin and improved cardiac function. Mechanistically, by lentiviral-based overexpression and CRISPR/Cas9-mediated silencing of Qki5 we identified regulated expression of specific circular RNAs derived from Ttn, Fhod3, and Strn3. Moreover, inhibition of Ttn derived circular RNA increased the susceptibility of cardiomyocytes to doxorubicin. We thus showed that overexpression of Qki5 strongly attenuates the toxic effect of doxorubicin via regulating a set of circular RNAs.
The first half of the ERC Consolidator grant was very successful. We have published a number of seminal papers in the field and provided the first expression patterns of cardiac lncRNAs both in cardiomyocytes (Science Transl Med. 2016 Feb 17;8(326):326ra22. doi: 10.1126/scitranslmed.aaf1475) and fibroblasts (Circ Res. 2017 Aug 18;121(5):575-583. ) during cardiac remodeling. Strikingly, we could develop a number of new treatment approaches based on the manipulation of cardiac lncRNAs (either by AAV-mediated overexpression or oligonucleotide-therapeutic silencing strategies). Our successful pilot study to create and use shRNA library screens to find functional relevant lncRNAs opens new doors for the future search of functional and thus targetable lncRNAs in cardiovascular diseases and beyond. In addition, several state of the art review articles about long noncoding RNAs have been published, which are highly cited and will provide a valuable source of information for a broad community of cardiovascular scientists (e.g. Circulation. 2016 Nov 8;134(19):1484-1499; Physiol Rev. 2016 Oct;96(4):1297-325.)
We have also made several public press releases to increase awareness about our research funded by the EU (e.g. https://www.mh-hannover.de/46.html?&L=1&tx_ttnews%5Btt_news%5D=4404&cHash=c1ed2f654cc3975e60ae2a477db165cd and News Section of our Homepage: https://www.mh-hannover.de/22300.html )
In addition, Prof. Thum organized a Keystone meeting (March 2017), where EU-Longheart funded projects have been presented. Also, ERC-funded junior researchers attended the Basic Science Summer school in 2017, organized by the European Society of Cardiology, at Sophia-Antipolis – France. Laura Santer was awarded the best poster prize for her poster presentation entitled ‘Circular RNAs for the therapy of cardiovascular disease’ which showcases the emergence of novel circular RNAs in the cardiovascular field. In March 2017, the Dr. Janika Viereck received the 1st poster prize during the 15th Dutch-German Joint Meeting of the Molecular Cardiology Groups. She has been awarded for her work on the development of a novel therapeutic strategy for the treatment of cardiac hypertrophy and heart failure based on AAV9-mediated delivery on lncRNA H19. In April 2017 the ERC junior researcher Maria-Teresa Piccoli won the 1st prize of the Young Investigator Award session “Heart Failure” at the 83rd annual meeting of the German Cardiac Society, for her talk titled “Inhibition of the cardiac fibroblast-enriched lncRNA Meg3 hinders MMP-2 induction following pressure overload ameliorating fibrosis and diastolic function”.