Periodic Reporting for period 4 - LONGHEART (Exploring selected long non-coding RNAs as diagnostics and therapeutic targets for heart failure)
Reporting period: 2019-12-01 to 2021-05-31
Despite clinical advances, diseases of the cardiovascular system are the most common cause of morbidity and mortality with currently up to 60 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. Mechanistically, cardiac stress such as myocardial infarction or hypertension leads to cellular “remodeling” of the left ventricle resulting in heart failure and death of the patient. Protein-coding genes originate from a minimal fraction 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). The overall objectives of this ERC Consolidator grant were to expand the knowledge from miRNAs to long noncoding RNAs and their role in cardiac remodeling. Specific objectives were 1) identify novel functional relevant cardiac remodeling-associated lncRNAs; 2) characterise key lncRNA cardiac targetomes; 3) investigate lncRNA-paracrine mechanisms and the diagnostic and prognostic potential of cardiac-derived extracellular lncRNAs using large clinical cohorts; and 4) discover their therapeutic potential to prevent cardiac remodeling in clinically relevant animal models. ERC LONGHEART led to ground-breaking new insight into the role of lncRNAs in the heart.
We established new lncRNA-based mechanisms and thus identified fundamentally novel diagnostic and therapeutic entry points for a most serious clinical important disorder in dire need for new diagnostic and therapeutic paradigms, heart failure.
We established new lncRNA-based mechanisms and thus identified fundamentally novel diagnostic and therapeutic entry points for a most serious clinical important disorder in dire need for new diagnostic and therapeutic paradigms, heart failure.
Within LONGHEART we focused 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 had the following interconnected objectives and I will briefly describe what we have done here in the 5-6 years:
1: identify novel functional relevant cardiac remodeling-associated lncRNAs;
We have implemented novel high-throughput methods for lncRNA identification and identified several lncRNA candidates as interesting novel therapeutic targets. For example lncRNA Chast (cardiac hypertrophy-associated transcript) (Science Transl Med. 2016;8:326ra22.) which triggers pathological growth and further remodelling processes in the heart, which ultimately lead to weakening of the heart function. Silencing of Chast both prevented and attenuated TAC (transverse aortic constriction)-induced pathological cardiac remodeling with no early signs on toxicological side effects. The lncRNA Meg3 (maternally expressed gene 3) was found to be a novel and innovative target in heart failure pathologies with fibrosis (Circ Res. 2017;121:575-583.). Besides the identification of several other lncRNAs we created a lentiviral shRNA library-based approach for functional lncRNA profiling (Cell Death Differ. 2018;25:307-318.). With this study we were able to demonstrate the applicability of an innovative shRNA library approach to identify long non-coding RNA functions in a massive parallel approach.
2: Characterisation of key lncRNA cardiac targetomes
Here, we showed that Chast interacts mainly with proteins involved in cardiomyopathies and correlates with cardiac disease pathways, qualifying Chast as a potential target for therapeutics (Science Transl Med. 2016;8:326ra22.). In a further study, it was shown that the lncRNA H19 is down-regulated in failing hearts from mice, pigs and humans (Eur Heart J, 2020 Sep 21;41(36):3462-3474). H19 acts as an anti-hypertrophic lncRNA and therefore represents a promising therapeutic target to combat pathological cardiac remodelling.
3: Investigation of lncRNA-paracrine mechanisms and the diagnostic and prognostic potential of cardiac-derived extracellular lncRNAs using large clinical cohorts
We discovered lncRNAs secreted by hypoxic cardiomyocytes. Specifically, we have unraveled a novel intercellular communication route between hypoxic cardiomyocytes and fibroblasts via extracellular vesicles, which are enriched with lncRNAs. Vesicles were taken up by fibroblasts, triggering expression of profibrotic genes. Genetic loss of a certain lncRNA in vivo resulted in an impaired heart function after myocardial infarction highlighting its translational relevance (Mol Ther Nucleic Acids, 2019;18:363-374).
4: Discover the therapeutic potential to prevent cardiac remodeling in clinically relevant animal models.
We have developed cell type specific targeting vectors for the modulation of cardiac lncRNAs and have provided first evidence for therapeutic manipulation of lncRNAs in heart diseases (Science Transl Med. 2016;8:326ra22). 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. (Circ Res. 2017 Aug 18;121(5):575-583). Finally, we reported on the powerful therapeutic potential of the conserved lncRNA H19 in the treatment of pathological cardiac hypertrophy. Pressure overload-induced cardiac hypertrophy in H19 knock-out mice was aggravated compared to wild-type mice. In contrast, vector-based, cardiomyocyte-directed gene therapy using murine and human H19 strongly attenuated heart failure even when cardiac hypertrophy was already established. H19 was highly conserved and down-regulated in failing hearts from mice, pigs and humans. We showed H19 gene therapy to prevent and reverse experimental pressure-overload-induced heart failure (Eur Heart J, 2020 ;41:3462-3474.).
Within the framework of the funding, many promising lncRNAs have been identified and functionally characterised and analyses have also been carried out with regard to their diagnostic and therapeutic potential. These were the objectives of the project and this was fully achieved.
1: identify novel functional relevant cardiac remodeling-associated lncRNAs;
We have implemented novel high-throughput methods for lncRNA identification and identified several lncRNA candidates as interesting novel therapeutic targets. For example lncRNA Chast (cardiac hypertrophy-associated transcript) (Science Transl Med. 2016;8:326ra22.) which triggers pathological growth and further remodelling processes in the heart, which ultimately lead to weakening of the heart function. Silencing of Chast both prevented and attenuated TAC (transverse aortic constriction)-induced pathological cardiac remodeling with no early signs on toxicological side effects. The lncRNA Meg3 (maternally expressed gene 3) was found to be a novel and innovative target in heart failure pathologies with fibrosis (Circ Res. 2017;121:575-583.). Besides the identification of several other lncRNAs we created a lentiviral shRNA library-based approach for functional lncRNA profiling (Cell Death Differ. 2018;25:307-318.). With this study we were able to demonstrate the applicability of an innovative shRNA library approach to identify long non-coding RNA functions in a massive parallel approach.
2: Characterisation of key lncRNA cardiac targetomes
Here, we showed that Chast interacts mainly with proteins involved in cardiomyopathies and correlates with cardiac disease pathways, qualifying Chast as a potential target for therapeutics (Science Transl Med. 2016;8:326ra22.). In a further study, it was shown that the lncRNA H19 is down-regulated in failing hearts from mice, pigs and humans (Eur Heart J, 2020 Sep 21;41(36):3462-3474). H19 acts as an anti-hypertrophic lncRNA and therefore represents a promising therapeutic target to combat pathological cardiac remodelling.
3: Investigation of lncRNA-paracrine mechanisms and the diagnostic and prognostic potential of cardiac-derived extracellular lncRNAs using large clinical cohorts
We discovered lncRNAs secreted by hypoxic cardiomyocytes. Specifically, we have unraveled a novel intercellular communication route between hypoxic cardiomyocytes and fibroblasts via extracellular vesicles, which are enriched with lncRNAs. Vesicles were taken up by fibroblasts, triggering expression of profibrotic genes. Genetic loss of a certain lncRNA in vivo resulted in an impaired heart function after myocardial infarction highlighting its translational relevance (Mol Ther Nucleic Acids, 2019;18:363-374).
4: Discover the therapeutic potential to prevent cardiac remodeling in clinically relevant animal models.
We have developed cell type specific targeting vectors for the modulation of cardiac lncRNAs and have provided first evidence for therapeutic manipulation of lncRNAs in heart diseases (Science Transl Med. 2016;8:326ra22). 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. (Circ Res. 2017 Aug 18;121(5):575-583). Finally, we reported on the powerful therapeutic potential of the conserved lncRNA H19 in the treatment of pathological cardiac hypertrophy. Pressure overload-induced cardiac hypertrophy in H19 knock-out mice was aggravated compared to wild-type mice. In contrast, vector-based, cardiomyocyte-directed gene therapy using murine and human H19 strongly attenuated heart failure even when cardiac hypertrophy was already established. H19 was highly conserved and down-regulated in failing hearts from mice, pigs and humans. We showed H19 gene therapy to prevent and reverse experimental pressure-overload-induced heart failure (Eur Heart J, 2020 ;41:3462-3474.).
Within the framework of the funding, many promising lncRNAs have been identified and functionally characterised and analyses have also been carried out with regard to their diagnostic and therapeutic potential. These were the objectives of the project and this was fully achieved.
We have published a number of seminal landmark papers in the field and provided the first expression patterns of cardiac lncRNAs both in cardiomyocytes (Science Transl Med. 2016;8:326ra22.) and fibroblasts (Circ Res. 2017;121:575-583. ) during cardiac remodeling. We could develop a number of completely 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 opened new doors for the future search of functional and thus targetable lncRNAs in cardiovascular diseases and beyond.
We have also made several public press releases to increase awareness about our research funded by the EU (e.g. https://www.mhh.de/presse https://www.item.fraunhofer.de/en/press-and-media/press-releases/fraunhofer-item-with-new-research-topics.html and News Section of our Homepage: https://www.mhh.de/institute-zentren-forschungseinrichtungen/imtts/test )
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, organized by the European Society of Cardiology, at Sophia-Antipolis – France. Many employees were awarded with best poster awards and later got superb next job offers in Universities such as Standford or the Industry.
We have also made several public press releases to increase awareness about our research funded by the EU (e.g. https://www.mhh.de/presse https://www.item.fraunhofer.de/en/press-and-media/press-releases/fraunhofer-item-with-new-research-topics.html and News Section of our Homepage: https://www.mhh.de/institute-zentren-forschungseinrichtungen/imtts/test )
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, organized by the European Society of Cardiology, at Sophia-Antipolis – France. Many employees were awarded with best poster awards and later got superb next job offers in Universities such as Standford or the Industry.