miRNA Overexpression Induces Cardiomyocyte Proliferation In Vivo
Authors: Andrew H Baker, Eva van Rooij
Journal: Molecular Therapy (2013)

Many millions of muscle cells in the heart are lost following a heart attack (myocardial infarction), and replacing or regenerating these cells is of fundamental importance for the long-term recovery of heart function. Therefore, any strategy that induces proliferation of adult cardiomyocytes in the region damaged by ischemic injury is of great interest in regenerative medicine. A major hurdle for replenishing damaged myocardium is that adult mammalian cardiomyocytes, unlike those of zebrafish, have a very limited proliferative capacity.1Jopling C Sleep E Raya M Marti M Raya A Belmonte JCI Zebrafish heart regeneration occurs by cardiomyocyte dedifferentiation and proliferation.Nature. 2010; 464: 606-609Crossref PubMed Scopus (957) Google Scholar,2Kikuchi K Holdway JE Werdich AA Anderson RM Fang Y Egnaczyk GF et al.Primary contribution to zebrafish heart regeneration by gata4+ cardiomyocytes.Nature. 2010; 464: 601-605Crossref PubMed Scopus (790) Google Scholar Indeed, a recent landmark study showed that the mouse heart loses the capacity to regenerate sufficient cells to recover from a myocardial infarction just a few days after birth.3Porrello ER Mahmoud AI Simpson E Hill JA Richardson JA Olson EN et al.Transient regenerative potential of the neonatal mouse heart.Science. 2011; 331: 1078-1080Crossref PubMed Scopus (1675) Google Scholar It is therefore very important to understand why the hearts of zebrafish and those of very young mice can completely regenerate after an infarction whereas those of the adult human heart cannot and instead form a scar that leads to compromised myocardial function. In a recent issue of Nature,4Eulalio A Mano M Ferro MD Zentilin L Sinagra G Zacchigna S et al.Functional screening identifies miRNAs inducing cardiac regeneration.Nature. 2012; (e-pub ahead of print 5 December 2012)PubMed Google Scholar Eulalio and colleagues shed some light on this issue by identifying a number of microRNA (miRNA) species that have the capacity to induce cardiomyocyte cell proliferation and improve heart function following myocardial infarction in rodents. Many therapeutic strategies have been attempted to overcome the fundamental physiological blocks that hinder regenerative medicine in the cardiovascular system. These include, but are not limited to, screening for small molecules that promote cardiomyocyte differentiation,5Willems E Spiering S Davidovics H Lanier M Xia Z Dawson M et al.Small-molecule inhibitors of the Wnt pathway potently promote cardiomyocytes from human embryonic stem cell–derived mesoderm.Circ Res. 2011; 109: 360-364Crossref PubMed Scopus (179) Google Scholar activation of resident cardiac stem cells,6Smart N Bollini S Dube KN Vieira JM Zhou B Davidson S et al.De novo cardiomyocytes from within the activated adult heart after injury.Nature. 2011; 474: 640-644Crossref PubMed Scopus (523) Google Scholar and delivery of cells of autologous, allogeneic, or pluripotent-derived sources.7Dimmeler S Zeiher AM Schneider MD Unchain my heart: the scientific foundations of cardiac repair.J Clin Invest. 2005; 115: 572-583Crossref PubMed Scopus (595) Google Scholar Although there has been a great deal of progress at the preclinical level, clinical data in well-controlled and sufficiently powered studies generally either are lacking or have not produced definitive findings.8Mohsin S Siddiqi S Collins B Sussman MA Empowering adult stem cells for myocardial regeneration.Circ Res. 2011; 109: 1415-1428Crossref PubMed Scopus (86) Google Scholar Therefore, further study of the signals that underlie proliferation of cells in the cardiovascular compartment is clearly warranted. Many recent studies have begun to unveil the expression pattern, function, and therapeutic potential of miRNAs in the cardiac system (reviewed in refs. 9Small EM Olson EN Pervasive roles of microRNAs in cardiovascular biology.Nature. 2011; 469: 336-342Crossref PubMed Scopus (988) Google Scholar and 10van Rooij E Olson EN MicroRNA therapeutics for cardiovascular disease: opportunities and obstacles.Nat Rev Drug Discov. 2012; 11: 860-872Crossref PubMed Scopus (498) Google Scholar). miRNAs are small, noncoding RNAs that exert powerful regulatory influences on gene expression by targeting sequences usually located in the 3′ untranslated region of a messenger RNA, leading to inhibition of its translation or its degradation. Eulalio et al. devised a strategy to identify miRNAs that influence the proliferation of cardiomyocytes in a specific fashion.4Eulalio A Mano M Ferro MD Zentilin L Sinagra G Zacchigna S et al.Functional screening identifies miRNAs inducing cardiac regeneration.Nature. 2012; (e-pub ahead of print 5 December 2012)PubMed Google Scholar Using high-throughput technology, they assessed the influence of nearly 1,000 miRNAs on the proliferation of neonatal rat cardiomyocytes. By transfecting cardiomyocytes with a library of miRNA mimics, they identified more than 200 miRNA sequences that promoted proliferation and more than 300 miRNAs that inhibited cell proliferation. Focusing on the sequences that stimulated proliferation, they further refined this list by performing a series of additional detailed in vitro experiments. Interestingly, they were able to cluster many miRNAs into their respective miRNA families through their ability to promote cell proliferation, including miRNAs that had previously been implicated in cardiomyocyte proliferation11Porrello ER Johnson BA Aurora AB Simpson E Nam YJ Matkovich SJ et al.MiR-15 family regulates postnatal mitotic arrest of cardiomyocytes.Circ Res. 2011; 109: 670-679Crossref PubMed Scopus (345) Google Scholar,12Porrello ER Mahmoud AI Simpson E Johnson BA Grinsfelder D Canseco D et al.Regulation of neonatal and adult mammalian heart regeneration by the miR-15 family.Proc Natl Acad Sci USA. 2013; 110: 187-192Crossref PubMed Scopus (521) Google Scholar,13Hullinger TG Montgomery RL Seto AG Dickinson BA Semus HM Lynch JM et al.Inhibition of miR-15 protects against cardiac ischemic injury.Circ Res. 2012; 110: 71-81Crossref PubMed Scopus (408) Google Scholar or induced pluripotent stem cell reprogramming.14Ryul Lee M Prasain N Chae HD Kim YJ Mantel C Yoder MC et al.Epigenetic regulation of NANOG by miR-302 cluster-MBD2 completes iPS cell reprogramming.Stem Cells. 2012; (published online 19 December 2012; dio: 10.1002/stem.1302)Google Scholar These comprehensive follow-up experiments, which enabled the authors to show that cytokinesis and cell cycle re-entry of postnatal cardiomyocytes was caused by increases in the levels of the specific miRNAs, led to the selection of miR-199a-3p and miR-590-3p as lead candidates for further evaluation. A major difficulty in understanding the function and translational potential of individual miRNAs is defining the gene targets and biological pathways that they affect. In this study, the authors used deep-sequencing data from cells that had been exposed to increased levels of the individual miRNAs and control cells so as to identify pathways and miRNA gene targets, including homer1, HOPX, and CLIC5, that contribute to the proliferative phenotype. These studies indicated, as one may expect, that effects on multiple genes and signaling pathways probably contribute to the observed efficacy. Clearly the profound effects observed on cell proliferation by simple manipulation of individual miRNAs suggest that each miRNA must act to regulate complex biological events leading to re-entry of cardiomyocytes into the cell cycle. Powerful in vivo experiments showed that ectopic overexpression of miR-199a-3p and miR-590-3p exerts beneficial effects on the mouse myocardium following infarction. Overexpression of either miRNA by viral delivery in the peri-infarcted region induced a reduction in infarct size that resulted in improved function and reduced remodeling. More thorough examination of the border-zone region showed the presence of more nuclei that are positive for the novel thymidine analog 5-ethynyl-2′-deoxyuridine (EdU), indicating an increase in proliferation. The authors also showed that the timing of miRNA expression is critical, in that benefit was achieved selectively when the miRNAs were injected immediately post infarction but not when administered several days later. Eulalio and colleagues’ paper presents a well-designed, unbiased screen, with subsequent analysis by miRNA manipulation in vitro and in vivo that allows for evaluation of translational potential. However, as is true for all novel, enticing findings, there are potential limitations that deserve noting. First, there is a relative lack of detail regarding the endogenous miRNA levels in cardiomyocytes in culture, if they are present at all, and the level of increase effected by miRNA mimicry. Second, transfection/infection probably results in a level of miRNA far higher than physiologically possible. Third, it will be important to learn more about the regulation and function of these miRNAs, particularly miR-199a-3p and miR-590-3p, using animal models and, where possible, human tissue. miR-199a-3p (formerly known as miR-199a*) is processed from the same stem loop precursor as miR-199a-5p (formerly miR-199a). Although some studies have defined contributions of miR-199a-5p to cardiac physiology and pathophysiology,15Song X-W Li Q Lin L Wang X-C Li D-F Wang G-K et al.MicroRNAs are dynamically regulated in hypertrophic hearts, and miR-199a is essential for the maintenance of cell size in cardiomyocytes.J Cell Physiol. 2010; 225: 437-443Crossref PubMed Scopus (117) Google Scholar,16Rane S He M Sayed D Vashistha H Malhotra A Sadoshima J et al.Downregulation of MiR-199a derepresses hypoxia-inducible factor-1α and sirtuin 1 and recapitulates hypoxia preconditioning in cardiac myocytes.Circ Res. 2009; 104: 879-886Crossref PubMed Scopus (504) Google Scholar,17Haghikia A Missol-Kolka E Tsikas D Venturini L Brundiers S Castoldi M et al.Signal transducer and activator of transcription 3–mediated regulation of miR-199a-5p links cardiomyocyte and endothelial cell function in the heart: a key role for ubiquitin-conjugating enzymes.Eur Heart J. 2011; 32: 1287-1297Crossref PubMed Scopus (105) Google Scholar relatively little is known about miR-199a-3p. For example, miR-199a-5p is a regulator of the hypoxic response in cardiomyocytes; downregulation of miR-199a-5p occurs at the posttranscriptional level but independent of regulation of miR-199a-3p (ref. 16Rane S He M Sayed D Vashistha H Malhotra A Sadoshima J et al.Downregulation of MiR-199a derepresses hypoxia-inducible factor-1α and sirtuin 1 and recapitulates hypoxia preconditioning in cardiac myocytes.Circ Res. 2009; 104: 879-886Crossref PubMed Scopus (504) Google Scholar). Furthermore, in the hypertrophied heart miR-199a-5p is upregulated, and overexpression in cardiomyocytes can increase cell size.15Song X-W Li Q Lin L Wang X-C Li D-F Wang G-K et al.MicroRNAs are dynamically regulated in hypertrophic hearts, and miR-199a is essential for the maintenance of cell size in cardiomyocytes.J Cell Physiol. 2010; 225: 437-443Crossref PubMed Scopus (117) Google Scholar,18van Rooij E Sutherland LB Liu N Williams AH McAnally J Gerard RD et al.A signature pattern of stress-responsive microRNAs that can evoke cardiac hypertrophy and heart failure.Proc Natl Acad Sci USA. 2006; 103: 18255-18260Crossref PubMed Scopus (1299) Google Scholar Although miR-199a-3p is expressed in cardiomyocytes, no studies before the one by Eulalio et al.4Eulalio A Mano M Ferro MD Zentilin L Sinagra G Zacchigna S et al.Functional screening identifies miRNAs inducing cardiac regeneration.Nature. 2012; (e-pub ahead of print 5 December 2012)PubMed Google Scholar have specifically focused on this miRNA in the heart. It will be of interest to study the cellular distribution and functions in other cell types given that miR-199a-3p has been shown to influence cancer cell invasion and cell-cycle kinetics,19Fornari F Milazzo M Chieco P Negrini M Calin GA Grazi GL et al.MiR-199a-3p regulates mTOR and c-Met to influence the doxorubicin sensitivity of human hepatocarcinoma cells.Cancer Res. 2010; 70: 5184-5193Crossref PubMed Scopus (357) Google Scholar,20Migliore C Petrelli A Ghiso E Corso S Capparuccia L Eramo A et al.MicroRNAs impair MET-mediated invasive growth.Cancer Res. 2008; 68: 10128-10136Crossref PubMed Scopus (156) Google Scholar and to promote proliferation and survival of endothelial cells, at least in culture.21Shatseva T Lee DY Deng Z Yang BB MicroRNA miR-199a-3p regulates cell proliferation and survival by targeting caveolin-2.J Cell Sci. 2011; 124: 2826-2836Crossref PubMed Scopus (132) Google Scholar The latter, in particular, could potentially suggest a functional effect in the vascular compartment that may be relevant to outcome in the cardiac setting. Further experiments could define the dose requirements for efficacy and determine the potential contribution of other cellular compartments to the beneficial effects observed on, for example, the vasculature or resident stem or progenitor cells. Defining the functional relevance of an miRNA can be problematic because of the often moderate regulation of several target genes. Detailed studies are probably required to decipher the series of events that lead to such a clear proliferative phenotype. Because the time course for the experiments presented is relatively short following infarction and miRNA delivery, it will be important to evaluate longer-term phenotypes and safety and, of course, to replicate such studies in larger models such as the rabbit or pig, which are well-recognized models of myocardial infarction that are more relevant to the pathophysiology of human myocardial infarction. The study by Eulalio et al. represents a major advance in that miRNAs have been identified that possess the capacity to induce postnatal cardiomyocyte cell cycle re-entry by impacting selected biological pathways. Although anti-miR studies have already shown great therapeutic potential to manipulate miRNA levels in vivo, oligonucleotide-based miRNA mimicry has been lagging behind.22van Rooij E Purcell AL Levin AA Developing microRNA therapeutics.Circ Res. 2012; 110: 496-507Crossref PubMed Scopus (418) Google Scholar With adeno-associated viruses already showing strong potential for cardiac gene delivery at the clinical level,23Jessup M Greenberg B Mancini D Cappola T Pauly DF Jaski B et al.Calcium upregulation by percutaneous administration of gene therapy in cardiac disease (CUPID): a phase 2 trial of intracoronary gene therapy of sarcoplasmic reticulum Ca2+-ATPase in patients with advanced heart failure.Circulation. 2011; 124: 304-313Crossref PubMed Scopus (570) Google Scholar the simplicity of the translational approach of overexpression of specific miRNAs to enhance cardiac regeneration is attractive. This, coupled with important miRNA-modulatory clinical trial data sets showing safety and potential efficacy (http://www.santaris.com/newsroom/news-releases/2011), suggests that the strategy described by Eulalio and colleagues may have important future therapeutic implications in cardiac gene therapy for patients who suffer heart attacks.