A new "lnc" between non-coding RNA and cardiac regeneration.

Myocardial infarction (MI) is a major threat to human health and is one of the most important diseases contributing to significant morbidity and mortality worldwide. Therapeutic strategies aimed at restoring cardiac reperfusion and improving cardiac regeneration after MI have been successful in preclinical models but have largely failed in clinical trials. In this context, a better understanding of the endogenous mechanisms of cardiovascular development is anticipated to lead to the identification of novel therapeutic factors. The recent findings demonstrating that both cardiovascular development and regeneration can be regulated by noncoding RNAs [such as microRNAs and long non-coding (lnc) RNAs] indicate the non-coding transcriptome as a potential key area to study for possible new options to promote cardiovascular regeneration in the setting of MI. In this issue of Cardiovascular Research, Li et al. suggest that loss of a newly identified lncRNA promotes endogenous cardiac regeneration after MI (Figure 1). The lncRNA, a non-coding splice variant of the AZIN2 gene (Ensembl ID: ENST00000497710.5), was identified as a potential cardiac lncRNA by comparing RNA sequencing of human foetal and adult hearts. The authors identified five potential novel lncRNAs that were differentially expressed and that correlated with cell cycle-related protein-coding genes, of which the non-coding AZIN2 splice variant (AZIN2-sv) was the most highly expressed in the heart. They confirmed that AZIN2-sv was conserved in rodents (mice and rats) and then tested the effects of adenovirus-mediated modulation of the transcript on 7day old (D7) and 1-day old (D1) rat cardiomyocytes and injured hearts. In mice, D1 and D7 are key time points in the context of cardiac regeneration. The neonatal mouse heart can fully regenerate after resection of the left ventricular apex, and after induction of MI at early stages (postnatal day 1, P1), however, this regenerative capacity is lost by 7 days of age, indicating the existence of a ‘regenerative window’ within the first week after birth. This regenerative capacity has been linked to the ability of cardiomyocytes to proliferate at P1 which is lost by P7. In the rat studies by Li et al., inhibition of AZIN2-sv in D7 cardiomyocytes improved their proliferation rate, whereas overexpression of the transcript in D1 cardiomyocytes significantly reduced proliferation. Similarly, in vivo inhibition of AZIN2-sv in D7 rat hearts, as well as in adult rat hearts after the induction of MI, was characterized by increased proliferation of cardiomyocytes. In contrast, overexpression of AZIN2-sv in D1 rat hearts was associated with reduced cardiomyocyte proliferation, thus confirming a role in regulating cardiomyocyte cell cycle. The outcome of the treated hearts was tested further, and loss of the transcript correlated with improved remodelling, reduced scarring, and elevated function as determined by serial echocardiography. lncRNAs can regulate a variety of biological functions, including chromatin remodelling, RNA stabilization and transcription regulation, as well as regulation of mRNA and microRNA (miRNA) expression. Li et al. investigated the mechanism of action of AZIN2-sv in cardiomyocytes and identified the miRNA-214/PTEN/Akt pathway as playing a critical role. MiRNA-214 has previously been shown to act on the PTEN/Akt pathway which in-turn has been implicated in cardiomyocyte survival, but this is the first evidence that this microRNA is regulated by a lncRNA. Interestingly, the expression of miRNA-214 inversely correlated with that of AZIN2-sv: miRNA-214 was highly expressed in the foetal heart but its expression decreased in the adult heart. MiRNA-214 is also one of the miRNAs that have been implicated in cardiovascular disease but its therapeutic potential still needs to be fully elucidated and it remains unknown whether miRNA-214 may have a protective or detrimental role in the disease-state. In this study, the fact that binding sites for AZIN2-sv within miRNA-214 were conserved between rat and human, suggests a potential therapeutic application of this lncRNA and its miRNA target in the setting of human MI. Another interesting observation by Li et al. is that AZIN2-sv inhibition did not affect cardiac fibroblasts but had a positive impact on post-MI angiogenesis in the infarcted rat heart (Figure 1). Importantly, angiogenesis has been shown to be beneficial in promoting healing and regeneration in the setting of ischaemic diseases, and the re-establishment of cardiac perfusion through angiogenesis has been proven to be critical for cardiac repair in experimental models of MI. Therefore, factors that are able not only to improve cardiomyocyte survival and proliferation but also angiogenesis and cardiac perfusion represent significant new therapeutic targets to enhance cardiovascular regeneration. That said, there are major limitations in considering lncRNAs for therapy such as the delivery and identification of specific downstream targets and biological effects. Further work to determine appropriate experimental delivery of lncRNA