In the past decade, studies have demonstrated that mammalian genomes produce thousands of long transcripts that have no or poor protein-coding potential. These transcripts are known as long non-coding RNAs (lncRNAs). Their expression can be dysregulated in various human disorders, including coronary heart disease. Only a small fraction of lncRNAs has been fully characterized. However, lncRNAs appear to be key modulators of cell fate and behavior. Several ongoing projects in our laboratories investigated the importance of lncRNAs in cardiac disease and regeneration.
We have recently demonstrated that a significant fraction of murine cardiac polyadenylated and multi-exonic lncRNAs are derived from developmental cardiac enhancers. Moreover, we have identified differentially modulated human lncRNAs during cardiac differentiation of isolated human fetal cardiac precursor cells. Many of these candidates are mapping to bona fide human fetal and adult cardiac enhancers, suggesting they represent enhancer-associated lncRNAs. We further characterized one of these candidate lncRNAs, which we named CARMEN. CARMEN is highly conserved in mouse, and appears to be an important regulator of cardiovascular cell specification and differentiation. Furthermore, CARMEN is derived from a human super-enhancer, which has been shown to be active in the adult heart.
In another project, we have annotated the mouse transcriptome after myocardial infarction via RNA sequencing and ab initio transcript reconstruction, and integrated genome-wide approaches to associate specific lncRNAs with developmental processes and physiological parameters. Expression of specific lncRNAs strongly correlated with defined parameters of cardiac dimensions and function. Using chromatin maps to infer lncRNA function, we identified many with potential roles in cardiogenesis and pathological remodeling. The vast majority was associated with active cardiac-specific enhancers. Importantly, oligonucleotide-mediated knockdown implicated novel lncRNAs in controlling expression of key regulatory proteins involved in cardiogenesis. Finally, we identified hundreds of human orthologues and demonstrate that particular candidates were differentially modulated in human heart disease.
A recent project investigates the importance of lncRNAs in cardiac disease, in particular in cardiac fibrosis. Using an integrated genomic screen, we identified Wisper (WIsp2 SuPer-Enhancer associated RNA) as a cardiac fibroblast-enriched lncRNA that regulates cardiac fibrosis after injury. Wisper expression was correlated with cardiac fibrosis both in a murine model of myocardial infarction and in heart tissue from human patients suffering with aortic stenosis. Loss of function approaches in vitro using modified antisense oligonucleotides demonstrated that Wisper is a specific regulator of cardiac fibroblast proliferation, migration and survival. Accordingly, silencing of Wisper in vivo attenuated myocardial infarction-induced fibrosis and cardiac dysfunction. Functionally, Wisper regulates cardiac fibroblast gene expression programs critical for cell identity, extracellular matrix deposition, proliferation and survival. In addition, it to control expression of a pro-fibrotic form of lysyl hydroxylase 2, implicated in collagen cross-linking and stabilization of the matrix. Together, our findings identify Wisper as a cardiac fibroblast-enriched super-enhancer associated lncRNA that represents an attractive therapeutic target to reduce pathological development of cardiac fibrosis in vivo.
Enhancers and long noncoding RNAs (lncRNAs) are key determinants of lineage specification during development. We are currently evaluating remodeling of the enhancer landscape and modulation of the lncRNA transcriptome during mesendoderm specification. Enhancer usage is coordinated with mesendoderm-specific expression of key lineage-determining transcription factors. Many of these enhancers are associated with the expression of lncRNAs. Importantly, examination of embryonic stem cell-specific enhancers interacting in three-dimensional space with mesendoderm-specifying transcription factor loci identified Meteor (MesEndoderm Transcriptional Enhancer Organizing Region). Genetic and epigenetic manipulation of the Meteor enhancer reveals its indispensable role during mesendoderm specification and subsequent cardiogenic differentiation via transcription-independent and dependent mechanisms. Loci similar to the Meteor locus, topologically associating a transcribed enhancer and its cognate protein coding gene, may represent a class of genomic elements controlling developmental competence in pluripotency.
Considering the promising results regarding lncRNAs, we decided to analyze RNA samples obtained from mouse hearts before and after myocardial infarction using RNA sequencing and ab initio reconstruction. We integrated genome-wide approaches to associate specific lncRNAs with developmental processes and physiological parameters. Expression of specific lncRNAs strongly correlated with defined parameters of cardiac dimensions and function. Using chromatin maps to infer lncRNA function, we identified many with potential roles in cardiogenesis and pathological remodeling. The vast majority were associated with active enhancers. Importantly, oligonucleotide-mediated knockdown implicated a novel lncRNA in controlling expression of key regulatory proteins involved in cardiogenesis. Finally, we identified hundreds of human orthologs. Particular candidates were differentially modulated in human heart disease. These findings reveal a novel class of heart-specific lncRNAs with unique regulatory and functional characteristics relevant to maladaptive remodeling, cardiac function and potentially cardiac regeneration.