miRNA Overexpression Induces Cardiomyocyte Proliferation In Vivo

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.^1,2 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.³ 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,⁴ 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,⁵ activation of resident cardiac stem cells,⁶ and delivery of cells of autologous, allogeneic, or pluripotent-derived sources.⁷ 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.⁸ 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. 9 and 10).

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.⁴ 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 proliferation^11,12,13 or induced pluripotent stem cell reprogramming.¹⁴ 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,^15,16,17 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. 16). Furthermore, in the hypertrophied heart miR-199a-5p is upregulated, and overexpression in cardiomyocytes can increase cell size.^15,18 Although miR-199a-3p is expressed in cardiomyocytes, no studies before the one by Eulalio et al.⁴ 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,^19,20 and to promote proliferation and survival of endothelial cells, at least in culture.²¹ 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.²² With adeno-associated viruses already showing strong potential for cardiac gene delivery at the clinical level,²³ 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.