A balancing act in cardiac hypertrophy

This editorial refers to ‘The H19 long noncoding RNA is a novel negative regulator of cardiomyocyte hypertrophy’ by L. Liu et al., pp. 56–65. Cardiac hypertrophy is the process of cardiac myocyte enlargement, typically in response to the need to generate greater levels of contractile force. One of the best examples of adaptive, non-pathological hypertrophy is the expansion of cardiac myocyte volume and sarcomere content that occurs during postnatal development. Initially, compensatory hypertrophy that occurs as a result of persistent hypertension or aortic stenosis can become maladaptive in the longer term, leading to alterations in myocyte electrical stability, sarcolemmal integrity, and potential myocyte loss. The molecular basis of hypertrophy initiation and progression has been well studied, with a host of genetic manipulations in model animal hearts used to define key chromatin remodelling events, alterations in transcription of coding and noncoding mRNA, and post-translational protein modifications required for execution of the hypertrophic programme. However, endogenous pathways that serve to limit the extent of hypertrophy have typically received less attention (Figure 1). Upper panel: summary of the previous literature demonstrating that H19 balances transcription of Igf2; less H19 leads to greater Igf2 levels. Lower panel: findings from Liu et al. (this issue) that in the mouse heart, H19 (acting primarily through generation of miR-675) serves to limit growth responses. Higher H19 and miR-675 levels decrease hypertrophy induction. In this issue of Cardiovascular Research, Liu et al.1 study the involvement of the H19 long noncoding RNA transcript in cardiac hypertrophy. The H19 locus is under imprinting control, with expression of H19 taking place exclusively from the maternal allele during the majority of embryonic development. H19 expression during embryogenesis limits generation of insulin-like growth factor 2 (IGF2) from a genomically adjacent region, and imprinting defects in this area that alter H19 levels can lead to overabundance of IGF2, promoting somatic overgrowth, or abnormally decreased IGF2, leading to somatic undergrowth.2 In humans, decreased H19 and increased IGF2 levels during development are a major cause of the Beckwith–Wiedemann syndrome (http://www.omim.org/entry/130650), while opposite H19/IGF2 dysregulation gives rise to the Silver–Russell syndrome (http://www.omim.org/entry/180860). In any case, H19 expression is markedly down-regulated immediately after birth in all tissues, except for the skeletal muscle.3 Contrary to embryonic regulation, levels of both H19 and Igf2 RNA transcripts decline in parallel during mouse cardiac postnatal development, measured from Postnatal Day 1 through to 5 weeks of age (RNAseq dataset NCBI GEO GSE689214) and similarly observed in the study of Liu et al.1 via quantitative reverse transcriptase–polymerase chain reaction (RT–qPCR). As for many noncoding RNAs, the mechanism by which H19 exerts its effects is not entirely clear. However, a more recent work than the initial H19-Igf2 characterizations has shown that the first exon of H19 encodes a stem-loop microRNA precursor; in this case, for miR-675; and naturally questions have arisen concerning the degree to which the production of this microRNA mediates H19 actions. Liu et al.1 show elevation of H19 in both their mouse transverse aortic constriction (TAC) model and phenylephrine (PE)-treated neonatal cardiomyocytes and observe that H19 is present both in adult cardiomyocytes and fibroblasts, although more abundant in the myocyte fraction. They do not assess whether Igf2 is altered in parallel to H19 at any stage, but instead focus on the role of miR-675 generated from its H19-embedded precursor. RT–qPCR assays of mature miR-675(-3p) show that its abundance changes in accordance with H19 alterations. Curiously, two independent miR-seq studies barely detected miR-675 in adult mouse hearts, finding at least 250 other microRNA species to be more abundant, although miR-675-3p was observed than miR-675-5p (NCBI GEO GSE35350, GSE55792).5,6 To explore the signals mediated by H19 and/or miR-675 during myocyte hypertrophy, the authors make extensive use of the neonatal cardiomyocyte model and a toolkit of reagents for overexpression and knockdown of H19 and for miR-675 specifically. The key finding is that both adenoviral transduction of H19 and myocyte transfection with miR-675 decrease basal and PE-induced hypertrophic markers and cell size, while the cognate siRNA and anti-miR are able to promote hypertrophy in the absence of PE. A mutant H19 construct lacking the miR-675 precursor is unable to replicate these effects. Thus, in vitro studies demonstrate that elevation of H19, and particularly of miR-675, by hypertrophic signals represents the simultaneous induction of a hypertrophy-limiting mechanism. The follow-up questions from such an observation are usually as follows: does the same effect hold true for intact hearts, and if so, which are the molecular entities that transduce the signal? Liu et al.1 demonstrate that an antagomiR against miR-675 injected via the tail vein is able to exacerbate TAC effects on heart weight, cell size, hypertrophic marker genes, and fibrosis. The usual caveat applies here, in that such antagomiR administration is not tissue or cell specific, leaving open the possibility of neurohumoral influences from other tissues in which miR-675 might be expressed. The absolute specificity of antagomiRs for their intended targets is also not certain, as demonstrated by competing findings with anti-miR-21 vs. a genetic knockout7,8 and the notion that downstream effects of microRNA inhibition may include dysregulated transcription of other microRNAs.9 To explore whether further pressure on the H19/miR-675 endogenous ‘brake’ against cardiac hypertrophy might be of therapeutic value, adeno-associated viri of the appropriate serotype,10 encoding H19 or just the miR-675 precursor, could be administered prior to or during TAC. Experiments of this kind, which increase cardiac specificity and are independent of potential ‘off-target’ effects of antagomiRs, will be important in either supporting or arguing against the findings of the current study. In terms of molecular mechanism, the authors take a traditional approach of using bioinformatic software to try to identify mRNAs with miR-675-3p ‘seed’-binding sequences and find that CaMKIIδ satisfies criteria for a miR-675-3p target in reporter and immunoblot assays in isolated cardiomyocytes, when miR-675 is manipulated with the same reagents used to demonstrate its involvement in PE hypertrophy. More importantly, in vivo antagomiR-675 leads to up-regulation of CaMKIIδ protein expression in the heart. These data represent strong circumstantial evidence gained from both in vitro and in vivo systems but still have the caveat that direct Ago-mediated miR-675 interaction with CaMKIIδ is not demonstrated. This is a technical obstacle which has only recently been cleared with the advent of cross-linking and Ago immunoprecipitation procedures (CLIP) coupled to RNA sequencing, and which still has not been demonstrated in the intact heart.11 Nonetheless, measurements of this kind are crucial to demonstrating that the effects of miR-675 on CaMKIIδ are truly direct in vivo, as well as to delineate what the other in vivo targets of cardiac miR-675 might be, in a high-throughput manner. Despite the above limitations, which are frankly shared by many studies in the field of cardiac microRNAs, the study by Liu et al.1 remains notable for reminding us that biological signalling is rarely a unidirectional process. At the same time that hypertrophy of the adult heart (or of post-embryonic cardiomyocytes) is initiated, another process with the intrinsic capacity to limit hypertrophy is also called into play. The decline of H19 during postnatal cardiac maturation occurs in parallel to the deceleration of postnatal cardiac enlargement, although it is of interest that exercised hearts (undergoing ‘physiological’ rather than TAC-induced ‘pathological’ hypertrophy) show decreased H19 in the current study. Additional studies of mechanistic effects downstream of H19/miR-675, and of the manner in which hypertrophic stimuli influence H19 transcription, should be instructive for determining whether amplified H19 signalling can restore balance in an inappropriately hypertrophying heart. Conflict of interest: none declared.