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Aims Production of functional cardiomyocytes from pluripotent stem cells requires tight control of the differentiation process. Long noncoding RNAs (lncRNAs) exert critical regulatory function 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 is 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 downregulation, MIR-133a2 target protein-coding genes are coordinately downregulated. 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 upregulated upon Notch1 silencing in ESCs. Forced expression of either linc1230 or linc1335 improved ESC-derived cardiomyocyte production. These two lncRNAs were also found upregulated 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 signaling pathway, for the coordinated regulation of cardiogenic differentiation in ESCs. Translational perspective Cardiac dysfunction and heart failure develop secondary to a massive loss of cardiomyocytes in the damaged myocardium. Several avenues have been evaluated to promote regeneration following injury. Cell therapy for heart disease envisages the production of functional cardiomyocytes from differentiating pluripotent stem cells prior transfer into the injured heart muscle. Here, we report the functional characterization of CARMA, a lncRNA implicated in cardiogenesis. CARMA knockdown in differentiating human embryonic stem cells (ESCs) promotes cardiogenic commitment and cardiomyocyte differentiation. CARMA represents therefore a novel target for improving human ESC-derived cardiomyocyte production, and cell-based regenerative strategies for heart disease.

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.

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., Long noncoding RNAs (lncRNAs) are key regulators of lineage specification during development. Here, the authors investigate remodeling of enhancers and regulation of the lncRNA transcriptome during mesendoderm specification, and identify a pluripotent stage-specific transcribed enhancer controlling adoption of the mesendodermal cell fate.

© 2014 . 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. •Fetal cardiac enhancers are transcribed generating enhancer derived lncRNAs.