VENTHEART is required for cardiomyocyte specification and function

Abstract Background Long noncoding RNAs (lncRNAs) control early stages of cardiac differentiation, however their role in later specification and maturation is still not well explored. Methods and results We performed single cell RNA-seq for 2, 6 and 12 week-old hESC-CM. Weighted correlation network analysis (WGCNA) identified core genes significantly upregulated, along with a subset of lncRNAs. Importantly, these lncRNAs are highly abundant and unique to human heart. Through independent integrative analysis of genome-wide association studies (GWAS) and expression quantitative trait locus (eQTL) data using human hearts, we also identified a long intergenic noncoding RNA (we call VENTHEART, VHRT) as co-regulated with core cardiac contractile genes, and strongly associated with heart failure. VHRT was highly expressed in MYL2+ hESC-CMs in our single cell dataset, and its locus is antisense and downstream of MYL2. VHRT knockdown (KD) in 6-weeks old hESC-CMs downregulated MYL2 and other key cardiac genes. Patch clamp recordings with VHRT KD cells showed a loss of the ventricular-like action potential. Concordantly, CRISPR-mediated excision of the VHRT locus led to impaired CM sarcomere formation, and loss of CM specification gene programs. VHRT transcript replacement in VHRT-KO cells was however insufficient to rescue the phenotype. Instead, we established by 3C assay, that the VHRT locus loops and interacts with the MYL2 promoter, bearing histone marks characteristic of a super-enhancer. Conclusion Thus, we conclude that both the VHRT lncRNA transcript and its genomic locus are required for proper CM specification and function, and may play a role in heart failure progression. Funding Acknowledgement Type of funding source: Other. Main funding source(s): EMBO, Singapore National Research Council