Genome-wide profiling of the cardiac transcriptome after myocardial infarction identifies novel heart-specific long non-coding RNAs.

AIM: Heart disease is recognized as a consequence of dysregulation of cardiac gene regulatory networks. Previously, unappreciated components of such networks are the long non-coding RNAs (lncRNAs). Their roles in the heart remain to be elucidated. Thus, this study aimed to systematically characterize the cardiac long non-coding transcriptome post-myocardial infarction and to elucidate their potential roles in cardiac homoeostasis. METHODS AND RESULTS: We 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 remodelling. 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. CONCLUSION: These findings reveal hundreds of novel heart-specific lncRNAs with unique regulatory and functional characteristics relevant to maladaptive remodelling, cardiac function and possibly cardiac regeneration. This new class of molecules represents potential therapeutic targets for cardiac disease. Furthermore, their exquisite correlation with cardiac physiology renders them attractive candidate biomarkers to be used in the clinic.

CARMEN, a human super enhancer-associated long noncoding RNA controlling cardiac specification, differentiation and homeostasis.

Long noncoding RNAs (lncRNAs) are emerging as important regulators of developmental pathways. However, their roles in human cardiac precursor cell (CPC) remain unexplored. To characterize the long noncoding transcriptome during human CPC cardiac differentiation, we profiled the lncRNA transcriptome in CPCs isolated from the human fetal heart and identified 570 lncRNAs that were modulated during cardiac differentiation. Many of these were associated with active cardiac enhancer and super enhancers (SE) with their expression being correlated with proximal cardiac genes. One of the most upregulated lncRNAs was a SE-associated lncRNA that was named CARMEN, (CAR)diac (M)esoderm (E)nhancer-associated (N)oncoding RNA. CARMEN exhibits RNA-dependent enhancing activity and is upstream of the cardiac mesoderm-specifying gene regulatory network. Interestingly, CARMEN interacts with SUZ12 and EZH2, two components of the polycomb repressive complex 2 (PRC2). We demonstrate that CARMEN knockdown inhibits cardiac specification and differentiation in cardiac precursor cells independently of MIR-143 and -145 expression, two microRNAs located proximal to the enhancer sequences. Importantly, CARMEN expression was activated during pathological remodeling in the mouse and human hearts, and was necessary for maintaining cardiac identity in differentiated cardiomyocytes. This study demonstrates therefore that CARMEN is a crucial regulator of cardiac cell differentiation and homeostasis.

Functional importance of cardiac enhancer-associated noncoding RNAs in heart development and disease.

The key information processing units within gene regulatory networks are enhancers. Enhancer activity is associated with the production of tissue-specific noncoding RNAs, yet the existence of such transcripts during cardiac development has not been established. Using an integrated genomic approach, we demonstrate that fetal cardiac enhancers generate long noncoding RNAs (lncRNAs) during cardiac differentiation and morphogenesis. Enhancer expression correlates with the emergence of active enhancer chromatin states, the initiation of RNA polymerase II at enhancer loci and expression of target genes. Orthologous human sequences are also transcribed in fetal human hearts and cardiac progenitor cells. Through a systematic bioinformatic analysis, we identified and characterized, for the first time, a catalog of lncRNAs that are expressed during embryonic stem cell differentiation into cardiomyocytes and associated with active cardiac enhancer sequences. RNA-sequencing demonstrates that many of these transcripts are polyadenylated, multi-exonic long noncoding RNAs. Moreover, knockdown of two enhancer-associated lncRNAs resulted in the specific downregulation of their predicted target genes. Interestingly, the reactivation of the fetal gene program, a hallmark of the stress response in the adult heart, is accompanied by increased expression of fetal cardiac enhancer transcripts. Altogether, these findings demonstrate that the activity of cardiac enhancers and expression of their target genes are associated with the production of enhancer-derived lncRNAs.

The conserved long non-coding RNA CARMA regulates cardiomyocyte differentiation.

AIMS: Production of functional cardiomyocytes from pluripotent stem cells requires tight control of the differentiation process. Long non-coding RNAs (lncRNAs) exert critical regulatory functions in cell specification during development. In this study, we designed an integrated approach to identify lncRNAs implicated in cardiogenesis in differentiating human embryonic stem cells (ESCs). METHODS AND RESULTS: We identified CARMA (CARdiomyocyte Maturation-Associated lncRNA), a conserved lncRNA controlling cardiomyocyte differentiation and maturation in human ESCs. CARMA is located adjacent to MIR-1-1HG, the host gene for two cardiogenic miRNAs: MIR1-1 and MIR-133a2, and transcribed in an antisense orientation. The expression of CARMA and the miRNAs are negatively correlated, and CARMA knockdown increases MIR1-1 and MIR-133a2 expression. In addition, CARMA possesses MIR-133a2 binding sites, suggesting the lncRNA could be also a target of miRNA action. Upon CARMA down-regulation, MIR-133a2 target protein-coding genes are coordinately down-regulated. Among those, we found RBPJ, the gene encoding the effector of the NOTCH pathway. NOTCH has been shown to control a binary cell fate decision between the mesoderm and the neuroectoderm lineages, and NOTCH inhibition leads to enhanced cardiomyocyte differentiation at the expense of neuroectodermal derivatives. Interestingly, two lncRNAs, linc1230 and linc1335, which are known repressors of neuroectodermal specification, were found up-regulated upon Notch1 silencing in ESCs. Forced expression of either linc1230 or linc1335 improved ESC-derived cardiomyocyte production. These two lncRNAs were also found up-regulated following CARMA knockdown in ESCs. CONCLUSIONS: Altogether, these data suggest the existence of a network, implicating three newly identified lncRNAs, the two myomirs MIR1-1 and MIR-133a2 and the NOTCH signalling pathway, for the coordinated regulation of cardiogenic differentiation in ESCs.

A transcribed enhancer dictates mesendoderm specification in pluripotency.

Enhancers and long noncoding RNAs (lncRNAs) are key determinants of lineage specification during development. Here, we evaluate remodeling of the enhancer landscape and modulation of the lncRNA transcriptome during mesendoderm specification. We sort mesendodermal progenitors from differentiating embryonic stem cells (ESCs) according to Eomes expression, and find that 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. Examination of ESC-specific enhancers interacting in three-dimensional space with mesendoderm-specifying transcription factor loci identifies MesEndoderm Transcriptional Enhancer Organizing Region (Meteor). Genetic and epigenetic manipulation of the Meteor enhancer reveal its indispensable role during mesendoderm specification and subsequent cardiogenic differentiation via transcription-independent and -dependent mechanisms. Interestingly, Meteor-deleted ESCs are epigenetically redirected towards neuroectodermal lineages. Loci, topologically associating a transcribed enhancer and its cognate protein coding gene, appear to represent therefore a class of genomic elements controlling developmental competence in pluripotency.