CARMEN-201, a specific isoform of an enhancer-associated long noncoding RNA controls smooth-muscle lineage specification in human cardiac precursor

Abstract

Background & Aim We have recently examined the cardiogenic potential of cardiac precursor cells (CPCs) isolated from the human fetal and the adult heart. The two populations of CPCs resemble mesenchymal stromal cells (expression of mesenchymal surface markers CD90, CD73, CD105) and cardiac transcription factors such as NKX2.5, GATA4, TBX5 and MEF2C. However, despite these phenotypical similarities, they behave very differentially upon differentiation in vitro. While fetal CPCs differentiate mainly into cardiomyocytes (CM), adult CPCs preferentially produce smooth muscle cells (SMC). We have previously demonstrated the importance of the enhancer-associated lncRNA CARMEN, in the specification and differentiation of fetal and adult CPCs. Several CARMEN isoforms have been described. We analyzed the role of the diffferent CARMEN isoforms in fetal and adult CPCs during differentiation into either CMs or SMCs. Methods, Results & Conclusion We found that the expression of one particular isoform, named CARMEN-201, the most induced during the differentiation of adult CPCs into SM, correlates with the expression of two miRNAs, MIR-143/145, located in the CARMEN locus. GapmeR-mediated depletion of CARMEN-201 results in a corresponding downregulation of the expression of the two miRNAs, and reduces the capacity of adult CPCs to produce SMCs. CARMEN-201 possesses a unique exon (Exon2) that can be targeted via CRISPR/Cas9 deletion. When adult CPCs are lacking this exon, their ability to differentiate into of SMCs is markedly reduced. In contrast, induction of CARMEN-201 expression in fetal CPCs using a CRISPR On approach is sufficient to trigger SMC differentiation. Similarly, lentiviral overexpression CARMEN-201 Exon2 redirects fetal CPCs into the SMC lineage. CARMEN-201 Exon2 displays a MIRc element. The capacity of fetal CPCs overexpressing CARMEN-201 Exon2 to differentiate into of SMCs is significantly declined when Exon2 is mutated in its MIRc element. We demonstrated that CARMEN-201 Exon2 physically interacts with RE1-silencing transcription factor (REST), NOP2/Sun RNA methyltransferase 6 (NSUN6) and replication protein A1 (RPA1). REST has been previously shown to play a crucial role in cardiac lineage specification. Our next goal is to determine the role of these proteins in SM linage specification. CARMEN-201 isoform represents a promising therapeutic target to regulate the production of CM or SMC in cell replacement therapies.