Micro-managing myocyte mitosis.

In the adult mammalian heart, cardiac myocyte renewal is rare1,2 and insufficient to restore normal pump function after significant myocardial damage. Recent studies, however, suggest that zebrafish hearts3–5 and neonatal mouse hearts6 can regenerate after injury through enhanced cardiac myocyte proliferation. In mice, this restorative potential is lost shortly after birth.6 In this issue of Circulation Research, Porrello et al identify the microRNA (miR)-15 family of miRs as potential mediators of the postnatal loss of proliferative potential.7 Most (if not all) adult mammalian cardiac myocytes permanently exit the cell cycle and do not proliferate even when challenged by injury or stress. Scientists have long sought to understand the mechanisms that prevent cardiac myocyte cell cycle reentry and to overcome these blocks to generate new functional myocytes.8 Experimental approaches in the past have included manipulation of direct cell cycle regulators such as cyclins and cyclin-dependent kinase inhibitors and regulation of myofibril disassembly, which is thought to be necessary for the mature cardiac myocyte to undergo cell division (cytokinesis).9 Excitement and enthusiasm for seeking a deeper understanding of this phenomenon have been enhanced by the discovery that adult zebrafish hearts can regenerate even after significant injury4 and by the more recent observation by Porrello et al that newborn mouse hearts can also regenerate.6 These findings indicate that functional cardiac myocytes, with contractile sarcomeres and myofibrils, are able to reenter the cell cycle and undergo cytokinesis to generate new myocytes. Importantly, this capacity is largely lost during the first week of postnatal life, suggesting that epigenetic changes during that time period alter the myocyte response to injury. Indeed, dramatic changes in gene expression occur shortly after birth as the fetal gene program is silenced and adult isoforms of contractile proteins, calcium transporters, and metabolic regulators are expressed.10 In rodents, most cardiac myocytes undergo 1 postnatal round of DNA synthesis without cytokinesis, resulting in binucleation and subsequent cell cycle arrest, usually at G0/G1.11 Altered response to injury, large-scale changes in gene expression, and uncoupling of cytokinesis from karyokinesis (nuclear division) that occur in the first week of life could be coordinately or separately regulated by 1 or more epigenetic modulators, and direct comparison of the presence or activity of candidate effectors in newborn and early postnatal hearts is an attractive approach for identifying relevant regulators. Porrello et al adopted this strategy to identify potential miRs that regulate cardiac myocyte proliferation.7 MiRs are short, noncoding RNAs that modulate stability or translation of mRNAs and may function to simultaneously target multiple members of a biological pathway. Indeed, several miRs have previously been implicated in the regulation of cardiac myocyte proliferation.12,13 By comparing miR expression in P10 and P1 murine hearts, Porrello et al found that miR-195 (a member of the miR-15 family) is significantly upregulated in P10 hearts and remains elevated throughout adulthood. Other members of the miR15 family are similarly regulated. Although detailed analysis by Porrello et al focused on the miR-15 family, it is worth noting that a total of 71 miRs were found to be altered (upregulated or downregulated) between P1 and P10 in this screen. To determine if members of the miR-15 family can function to regulate cardiac myocyte proliferation, gainand loss-of-function studies were performed. Transgenic mice overexpressing miR-195, using the ß-myosin heavy chain promoter, exhibit cardiac myocyte hypoplasia and ventricular septal defects. At P1, the number of myocytes undergoing mitosis is decreased 3-fold (as assessed by phospho-histone H3 staining) and the number of multinucleated cells is increased. Microarray analysis suggests that mitosis and cell cycle genes are repressed both in vivo (in transgenic hearts) and in cultured myocytes infected with an adenovirus expressing miR-195. The pattern of gene expression is consistent with the induction of a G2/M arrest. Although cardiac-specific genetic inactivation (knockout) of the miR-15 family is not reported in this study, the function of these miRs was disrupted by the use of locked-nucleic acid–modified oligonucleotides directed against miR-15b and miR-16 delivered by subcutaneous injection. Administration of these long-lived inhibitory oligonucleotides once daily from P2-P4 appears to be sufficient to repress all miR-15 family members until at least P12. A significant 3-fold increase in phospho-H3-stained myocytes is detected after miR-15 inhibition, although evidence for cardiac myocyte cell division is not reported and cardiac myocytes expressing Aurora B kinase, which is necessary for cytokinesis, were not detected. These results suggest that the miR-15 family is likely to regulate important aspects of postnatal withdrawal from cell cycle progression in the heart but that inhibition of the miR-15 family is not sufficient to induce cytokinesis in adult myocytes. It will be interesting to determine if the response to injury in the adult heart is altered by inhibition or The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association. From the Department of Cell and Developmental Biology, the Cardiovascular Institute, and the Institute for Regenerative Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA. Correspondence to Jonathan A. Epstein, MD, 1154 BRB II, 421 Curie Blvd, Philadelphia, PA 19104. E-mail [email protected] (Circ Res. 2011;109:611-613.) © 2011 American Heart Association, Inc.