Extensive research has focused on the identification of stem and progenitor cells able to differentiate into cardiomyocytes after injury, and recent findings in zebrafish offer hope that heart regeneration could be induced in mammals. In this article, we examine the current knowledge concerning the mechanisms underlying heart regeneration.
Over the last few years, extensive research has demonstrated that the mammalian heart is unable to regenerate lost contractile tissue based on two major observations: first, the restricted proliferative potential of cardiomyocytes and cardiac progenitor cells; second, the low number of progenitor cells present in the adult heart. In the last few years, at least three cell populations that have the potential to generate new cardiomyocytes postnatally have been identified Citation[1–3]. However, it is still a debate as to why they fail to support natural regeneration after ischemic infarct or under other pathologic conditions. Indeed, a recent study indicates that the c-kit+ progenitor population does not, in fact, possess any myogenic potential Citation[4].
In contrast to mammals, a number of nonmammalian vertebrate species are able to regenerate their heart Citation[5–7], including the zebrafish Citation[8,9], which can fully regenerate its heart following amputation of up to 20% of the ventricle. To directly identify the source of newly formed cardiomyocytes during zebrafish heart regeneration, our group and others established a genetic strategy to lineage-trace cardiomyocytes in the adult fish, based on the cre/lox system widely used in the mouse Citation[10,11]. Using this approach, both groups found that heart regeneration in zebrafish is driven by pre-existing cardiomyocytes, rather than by progenitor cells.
To establish this finding, the authors first generated zebrafish transgenic lines in which tamoxifen-inducible cre recombinase is under the control of cardiomyocyte-specific promoters (mlc2a and gata4, respectively), which allow cardiomyocytes to be genetically labeled with green fluorescent protein (GFP) after excision of a loxP-flanked stop cassette. If these GFP-labeled fish are subsequently amputated and allowed to regenerate, the source of the new cardiomyocytes can be determined by simply looking at whether or not they express GFP. No GFP signal would indicate that the new cardiomyocytes have come from a source of progenitor cells, and GFP-labeled cells would indicate that the new cardiomyocytes have arisen from the proliferation of existing GFP-positive cardiomyoctes. Both groups showed conclusively that the majority, if not all, of the newly formed cardiomyocytes were GFP-positive and as such must have come from existing GFP-labeled cardiomyocytes. Moreover, 5-bromodeoxyuridine (BrdU) labeling assays demonstrated that a significant increase in the number of BrdU/GFP-positive cardiomyocytes takes place in regenerating hearts compared with nonamputated controls. From these data, the authors concluded that differentiated GFP-positive cardiomyocytes had re-entered the cell cycle.
However, this regenerative ability, described so far in zebrafish or adult newt, is not present in the mammalian heart, where cardiomyocytes withdraw from the cell cycle soon after birth, and subsequent growth after damage relies mainly on cellular hypertrophy and proliferation of other cell types. Last year, scientists attempted to determine whether mammalian cardiomyocytes show any proliferative potential. Pursuing this question, Bersell and colleagues identified neuregulin-1 as a major factor able to induce cell cycle re-entry and repair of heart injury in rats Citation[12]. Moreover, the authors also demonstrated that differentiated rat cardiomyocytes disassemble their sarcomeres in the midzone during karyokinesis and cytokinesis (ultimate steps of the mitotic cell cycle). Interestingly, we have described a similar process occurring during zebrafish heart regeneration Citation[10]. Electron microscopy of regenerating cardiomyocytes clearly showed that the sarcomeric structure has been disassembled and the cells have detached from one another to allow them to divide. This is also in line with recent microarray data of zebrafish heart regeneration, which show that sarcomeric genes are downregulated during these processes. Furthermore, this is accompanied by an increase in the expression of the cell cycle regulator plk1, inhibition of which effectively blocks regeneration. Similar events have also been described in the newt, where cardiac sarcomeric genes are downregulated after heart amputation Citation[13,14]. Although there is no evidence that mammalian cardiomyocytes can regenerate, such changes in structure and gene expression have also been observed in hibernating cardiomyocytes in humans after cardiac injury. It is therefore tempting to speculate that these cardiomyocytes are in fact attempting to proliferate Citation[11].
However, since the discovery of stem and progenitor cells that are able to differentiate in vitro into mature and functional cardiomyocytes, the field of cardiovascular medicine is pushing research to develop strategies that improve replacement of damaged cells with stem cells in order to mediate functional repair. Unfortunately, cell-based therapies for cardiovascular disorders need to address many important and limiting factors. One of the most remarkable problems is the insufficient homing and survival of transplanted cells in the ischemic scenario, together with the absence of cardiomyogenesis Citation[15]. This is in contrast to the evidence provided from the previously cited publications in newt, zebrafish and rat, where it has been demonstrated that cardiomyocytes with proliferative potential exist. In fact, in rats approximately 50% of mononucleated cardiomyocytes that re-enter the cell cycle can complete cytokinesis Citation[12].
Thus, if the capacity for cardiac regeneration is an ancestral condition that has been attenuated in the course of vertebrate evolution Citation[16], a complete understanding of cardiomyocyte cell cycle regulation is crucial. Defining the molecular basis of regeneration from mammalian vertebrates will be necessary for the field of mammalian cardiac regeneration. In the same direction, although several cardiac progenitors have been identified in the last decade, it is evident that their limited proliferation potential represents another problem in a therapeutic scenario. Research will not only have to be focused on the identification of these populations, but also on their activation, expansion and differentiation into functional cardiomyocytes. Finally, the existence of common repair mechanisms among species should encourage us to further investigate the molecular basis of cardiomyocyte cell cycle regulation after injury.
Financial & competing interests disclosure
Work in the laboratory of Juan Carlos Izpisúa Belmonte was supported by grants from MICINN, TERCEL and Fundacion Cellex. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
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