Long noncoding RNA VENTHEART is required for cardiomyocyte specification and function

Abstract

RationaleLong noncoding RNAs (lncRNAs) control cardiac gene expression during heart development and disease. It is accordingly plausible for the same lncRNA to regulate both cardiac development, as well as play a role in adult heart disease progression. lncRNA regulators of early cardiomyocyte (CM) lineage commitment have been identified and characterised, however those controlling later CM specification remain unknown. ObjectivesIn this study we identified a novel lncRNA required for CM specification, maturation and function, and also discovered its suggested relevance to heart disease. Methods and ResultsWe performed single cell RNA-seq on human embryonic stem cell derived cardiomyocytes at 2, 6 and 12 weeks of differentiation. Weighted correlation network analysis (WGCNA) identified core gene modules, including lncRNAs highly abundant and uniquely expressed in the human heart. A lncRNA (we call VENTHEART, VHRT) co-expressed with cardiac maturation and ventricular-specific genes MYL2 and MYH7, as well as in adult human ventricular tissue. CRISPR-mediated excision of VHRT led to impaired CM sarcomere formation, and loss of the CM specification gene program. VHRT knockdown (KD) in hESC-CMs confirmed its regulatory role for key cardiac contraction, calcium hemostasis and heart development genes, including MYH6 and RYR2. Functional evaluation after VHRT KD using impedance-based technology and action potential recordings, proved reduced contraction amplitude and loss of the ventricular-like action potential in CM, respectively. Through an integrative analysis of genome-wide association studies (GWAS), expression quantitative trait locus (eQTL) and gene co-expression network, we found VHRT to be co-regulated with core cardiac contractile genes, and the likely source of a heart failure genetic association signal overlapping the VHRT gene locus. Finally, VHRT KD and human failing heart transcriptome comparison validates the consistent downregulation again of cardiac contractile and calcium regulatory genes (P<0.05). ConclusionWe conclude that VHRT lncRNA is required for proper CM specification and function. Furthermore, reduced VHRT may contribute to the development or progression of human heart disease.