RNA mimics as therapeutics for cardiac regeneration: A paradigm shift

Through an elegant combination of transgenic techniques, a study by Tian et al. recently published in Science Translational Medicine1 shows that a cluster of five microRNAs (miRNAs), miR-302/367, controls cardiac myocyte proliferation during embryonic and neonatal life. More notably, reactivation of these miRNAs following myocardial infarction promotes re-entry of adult cardiomyocytes into the cell cycle and induces cardiac regeneration. The authors found that these miRNAs interfere with the recently described Hippo pathway that regulates proliferation of various cell types, including cardiomyocytes. More specifically, miR-302/367 directly reduces the expression of three inhibitory proteins in this pathway, leading to the activation of the Yap1 transcription factor, a master regulator of cardiac proliferation. The notion that miRNAs control cardiomyocyte proliferation is not entirely novel. Over the past few years, there has been a flood of exciting evidence that the miRNA network plays an essential role in regulating the extent of cardiomyocyte replication during development and that it might be harnessed in adult hearts to promote repair after myocardial damage. The sudden—and still, for the most part, not understood—arrest of cardiomyocyte proliferation immediately after birth correlates with increased levels of a large set of miRNAs, some of which are causally involved in this event. These include the six members of the miR-15 family2 and miR-29a,3 which target essential components of the cell cycle and checkpoint machineries; inhibition of miR-15 expands the proliferative potential of cardiomyocytes during fetal and early postnatal life and stimulates regeneration after myocardial infarction.4,5 By contrast, several miRNAs act in an opposite manner, by stimulating cardiomyocyte proliferation. A genomic-scale, high-throughput screen performed in my laboratory has shown that at least 40 miRNAs encoded by the human genome are capable of stimulating proliferation of neonatal cardiomyocytes.6 Among these miRNAs are miR-199a-3p, miR-590–3p, and two large families of miRNAs that previous work had shown to be essential in the regulation of embryonic cell proliferation: the miR-17/92 and the miR-302/367 clusters.7 Members of both families are highly expressed in embryonic stem cells and are essential to maintain the undifferentiated state.8 Transgenic overexpression of both the miR-17/92 cluster9 and the miR-302/367 cluster, as shown by Tian et al.,1 induces significant expansion of the cardiomyocyte pool during embryonic life, whereas their knockout leads to hypoplasia and cardiac dysfunction. More notably, in both cases reactivation of cardiomyocyte proliferation in adult mice is capable of increasing the usually ineffectual attempt at cardiac regeneration that is observed after myocardial infarction. The results of the new study strengthen and significantly expand a paradigm shift that has been occurring in the cardiovascular field over the last few years, namely that cardiac regeneration after damage can be achieved by stimulating the proliferation of already existing cardiomyocytes rather than having to rely on the implantation of exogenous cells. The notion that cardiomyocytes, or at least a subset of these, are endowed with an intrinsic capacity to proliferate during the adult life is consistent with earlier observations that a limited regenerative attempt normally occurs after infarction,10 that almost 50% of the myocardium is recycled during a lifetime,11 and that cardiac cell turnover physiologically occurs at a magnitude of ~1% cells per year.12 Expanding the endogenous cardiomyocyte proliferation capacity by genetic drugs thus appears to represent an exciting and more sustainable approach to achieve cardiac regeneration compared to ex vivo expansion and implantation of stem cell–derived cardiomyocytes. In this respect, it is worth recalling that cardiac regeneration in zebrafish, which occurs through the partial dedifferentiation and proliferation of preexisting cardiomyocytes,13,14 is also regulated by the miRNA network. In particular, recent evidence shows that miR-99/100 and Let-7a/c are downregulated during the regeneration process in zebrafish and that their forced inhibition is also effective in promoting regeneration in the mouse heart.15 Another merit of the article by Tian et al. is the description of a molecular mechanism for the miR-302/367 proliferative action based on its effect on the Hippo pathway. Originally discovered by genetic screens in the Drosophila eye, this pathway is a broad and essential regulator of cell proliferation and organ size. In mammalian cells, the main effector of the pathway is the transcriptional coactivator Yap. The kinase Mst1/2 (Hippo in Drosophila) interacts with WW45 to phosphorylate and activate the Lats1/2 and Mob1 complex, which in turn phosphorylates and inactivates Yap. In a crescendo of findings over the past few years, different investigators have shown that embryonic knockout of Mst1, WW45, and Lats causes cardiac hyperplasia, whereas overexpression of Mst1 and Lats, or knockout of Yap, leads to hypoplasia and cardiac dilation (reviewed in ref. 16). The pathway also appears to be active during postnatal life: in transgenic mice that overexpress a constitutively active Yap mutant, myocardial infarction is repaired with reduced fibrosis and increased myocardial tissue formation;17 consistent with this, Hippo deficiency enhances cardiomyocyte generation.18 Thus, the Hippo pathway stands as a master regulator of cardiac cell proliferation during both embryogenesis and postnatal life. The new study strengthens this conclusion by showing that at least one of the mechanisms of action of the miR-302/367 cluster is through repression of the kinases Mst1 and Lats2/Mob1b. Whether other miRNAs with the capacity to stimulate cardiomyocyte proliferation also act through the Hippo pathway remains a very interesting question that deserves further investigation. A most striking finding by Tian et al. is the demonstration that cardiac regeneration after myocardial infarction could be achieved by the administration, once a day, of synthetic miRNA mimics in a neutral lipid formulation. This finding is truly groundbreaking because, in the field of small-RNA therapeutics, it was thought that miRNA mimics would find little therapeutic usefulness because of their short half-life. This misconception arises because miRNA mimics, in contrast to miRNA inhibitors, must maintain their native chemical structure so as to be properly recognized by the RNA interference machinery, and thus could not be chemically modified to increase stability. The finding that the miR302b/c mimics are effective following systemic administration paves the way to a number of other mimic applications within and beyond the cardiovascular field, without the need to rely on viral vectors for intracellular miRNA expression. Will the miR302b/c mimics themselves become a real drug for patients with myocardial infarction? This is difficult to predict at this moment, but the biological activity of this miRNA cluster in inducing massive dedifferentiation toward an embryonic stem cell phenotype,19 and thus its deleterious effects when expressed for prolonged periods, would suggest caution. Should other miRNAs be identified that are equally effective in acting through the Hippo pathway but less prone to induce massive cell dedifferentiation, these would probably be better suited to development into human therapeutics. Despite this caveat, Tian et al. provide formidable proof-of-concept evidence that organ regeneration in vivo can be attained by exogenously delivered small-RNA drugs—certainly a welcome possibility, given the current critical burden of degenerative diseases.