Getting to the heart of the matter: long non-coding RNAs in cardiac development and disease

Abstract

Cardiogenesis in mammals requires exquisite control of gene expression and faulty regulation of transcriptional programs underpins congenital heart disease (CHD), the most common defect among live births. Similarly, many adult cardiac diseases involve transcriptional changes and sometimes have a developmental basis. Long non-coding RNAs (lncRNAs) are a novel class of transcripts that regulate cellular processes by controlling gene expression; however, detailed insights into their biological and mechanistic functions are only beginning to emerge. Here, we discuss recent findings suggesting that lncRNAs are important factors in regulation of mammalian cardiogenesis and in the pathogenesis of CHD as well as adult cardiac disease. We also outline potential methodological and conceptual considerations for future studies of lncRNAs in the heart and other contexts.

Figure 1

LncRNAs are a heterogeneous class of transcripts and function to regulate gene expression by diverse mechanisms. (A) Representative classes of long non-coding RNAs based on genomic location. LncRNAs can be located and transcribed within introns of protein-coding genes (left), as intervening genes known as long intergenic or intervening non-coding RNAs (lincRNAs) that do not overlap with the exons of other genes (middle), or they can be located on the opposite strand of a coding or non-coding gene and transcribed in the antisense direction (right). (B) Global mechanisms of lncRNA function. LncRNAs can function as molecular scaffolds by interacting with proteins such as transcription factors or components of chromatin modifying complexes to affect positive or negative regulation of gene expression (left panel). Proposed mechanisms of action include targeting proteins to specific genomic sites such as promoter regions by complementary interactions with DNA. Alternatively, interactions with DNA may prevent the binding of specific factors to the DNA template (middle). LncRNAs can also base pair with other RNA molecules such as mRNAs or may act as a sponge for miRNAs. This scenario is thought to lead to post-transcriptional gene silencing (right panel).

Figure 2

The developmental complexity of heart development varies among organisms despite conserved core cardiac TF network. (A) Drosophila heart development begins during embryonic stage 11 by specification of two contralateral rows of cardiogenic mesoderm and formation of cardioblasts. Cardioblasts migrate towards the midline at stage 13–14 and form a simple linear closed tube with a central lumen by stage 16–17, subsequently differentiating into more mature cardiomyocytes. (B) The first steps of mammalian heart development proceed in a very similar manner, yet the mature heart is considerably more complex with two atrial and two ventricular chambers, connecting the systemic and pulmonary circuits via four valves and in- and outgoing vessels. The earliest step of mammalian cardiogenesis involves the bilateral specification of cardiac progenitor cell populations from the first heart field (FHF) in the anterior lateral plate mesoderm, which condense into two lateral heart primordia (mouse E7.5, human day 15) to form the cardiac crescent. The secondary heart field (SHF) constitutes a separate cell population at the medial sides of the two processes of the cardiac crescent. The two processes of the cardiac crescent fuse to form a beating primitive linear heart tube (mouse E8.5, human day 21), which then undergoes rightward looping, resulting in formation of the early chambers (mouse E9, human day 28). During later stages, the mature shape of the heart is generated by differentiation of cardiac cell populations and extensive remodelling of the heart, resulting in four-chambered heart with distinct in- and outflow tracts, cardiac valves separating the different compartments, and a mature conduction system. (C) The core transcription factor network necessary for specification of the cardiovascular lineages is conserved between Drosophila and mammals. (D) The percentage of non-coding to protein-coding sequence increases with developmental complexity. Whereas S. cerevisiae dedicates most of its genome to protein-coding genes, only a small fraction of the genome codes for proteins in human. (E) The total number of putative lncRNA transcripts is predicted to be significantly higher in mouse (∼3000 lincRNA transcripts as determined by Ponjavic et al, 2007 and Sigova et al, 2013) and human (∼15 000 as defined by Derrien et al, 2012) as compared to lower eukaryotes such as Drosophila (17 based on stringent criteria in Tupy et al, 2005 to greater than 1000 based on low stringency estimates from Young et al, 2012), C. elegans (262 from Nam and Bartel, 2012), and zebrafish (∼700 transcripts predicted from Ultisky et al, 2011 and Pauli et al, 2012). The number of lncRNAs varies among studies as different criteria were used to define lncRNA transcripts.

Figure 3

Mechanisms of lncRNA function in heart development and cardiac disease. (A) Braveheart is necessary for commitment to the cardiac lineage in mouse. Bvht appears to function in trans through interaction with the epigenetic silencing complex PRC2 and may act as a decoy to antagonize its recruitment to key developmental genes during cardiomyocyte differentiation. Alternatively, Bvht may recruit PRC2 to gene(s) that repress the cardiac program. In either case, loss of Bvht leads to a failure to activate the core cardiac gene network that includes many TFs implicated in heart development and disease. (B) Fendrr is expressed in the lateral plate mesoderm in mouse from which precursors for the heart and body wall are derived. Fendrr is proposed to function partly in cis to regulate its neighbouring gene Foxf1a. Fendrr also functions in trans to regulate the expression of additional genes important for heart development. Fendrr interacted with PRC2 components as well as WDR5, a member of TrxG/MLL complex suggesting that Fendrr regulates the balance between repressive and activating marks at key genes during development. Thus, Bvht and Fendrr may represent examples of lncRNAs that regulate gene expression through epigenetic mechanisms. (C–E) LncRNAs can also function as natural antisense transcripts (NATs) to affect gene expression at the transcriptional and post-transcriptional level. ANRIL was identified as a risk factor for coronary disease by GWAS. ANRIL is expressed in the opposite direction to INK4B/P15 in the INK4 locus. The antisense transcript appears to recruit PRC1 and PRC2 to mediate repression of the INK4a/INK4b tumour suppressor locus through an epigenetic silencing mechanism (C). The ratio of two important sarcomere components MYH6 and MYH7 vary during development and in stress-induced pathological conditions. Myh6 and Myh7 genes are juxtaposed in the mouse genome in a head-to-tail fashion. An antisense lncRNA (Myh7-as) is transcribed across the Myh7 locus and negatively correlates with MYH7 abundance. Thus, Myh7-as transcription may regulate the ratio of Myh6 and Myh7 (D). Some antisense transcripts are predicted to form RNA duplexes with their mRNA counterpart leading to post-transcriptional regulation of the target message. For example, antisense transcripts to Alc1 and cNTI, two genes that code for important sarcomere components in cardiac muscle, form RNA duplexes with the respective protein-coding transcript. Alc1 antisense is increased in hypertrophic ventricles in patients with Tetralogy of Fallot, whereas elevated cTNI levels are correlated with ischaemia and risk of heart failure. In both of these cases, antisense transcripts may be important for regulating gene expression through formation of RNA duplexes that are substrates for recruitment of factors that degrade the mRNA or that physically block translation of the message. RNA–RNA interactions can also stabilize the mRNA in some cases (E).