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The HeArt of Regeneration

2006; Cell Press; Volume: 127; Issue: 3 Linguagem: Inglês

10.1016/j.cell.2006.10.025

1097-4172

Silvia Curado, Didier Y. R. Stainier,

Congenital heart defects research

Fish and amphibian hearts are known to regenerate after partial resection, but the molecular mechanisms underlying this process remain unclear. In this issue of Cell, Lepilina et al., 2006Lepilina A. Coon A.N. Kikuchi K. Holdway J.E. Roberts R.W. Burns C.G. Poss K.D. Cell. 2006; (this issue)Google Scholar analyze regeneration in the zebrafish heart. Their work indicates that new cardiomyocytes originate from undifferentiated progenitor cells and reveals a critical role for the epicardium, the cellular layer that covers the heart. Fish and amphibian hearts are known to regenerate after partial resection, but the molecular mechanisms underlying this process remain unclear. In this issue of Cell, Lepilina et al., 2006Lepilina A. Coon A.N. Kikuchi K. Holdway J.E. Roberts R.W. Burns C.G. Poss K.D. Cell. 2006; (this issue)Google Scholar analyze regeneration in the zebrafish heart. Their work indicates that new cardiomyocytes originate from undifferentiated progenitor cells and reveals a critical role for the epicardium, the cellular layer that covers the heart. Injury to the myocardium is a major cause of death, as the human heart has a limited capacity to regenerate. Possible approaches to treat heart failure include (1) transplantation of bone marrow or other progenitor cells into the heart and (2) boosting regeneration through inducing endogenous cells to differentiate/proliferate in situ to replace lost cardiomyocytes. Recent clinical trials injecting bone marrow into the injured heart have yielded mixed results (reviewed by Rosenzweig, 2006Rosenzweig A. N. Engl. J. Med. 2006; 355: 1274-1277Crossref PubMed Scopus (322) Google Scholar). Manipulating the regeneration potential of the adult heart may be the best strategy, but it is also the most challenging. Adult zebrafish, in contrast to mammals, are able to fully regenerate cardiac muscle (Poss et al., 2002Poss K.D. Wilson L.G. Keating M.T. Science. 2002; 298: 2188-2190Crossref PubMed Scopus (1236) Google Scholar). Following surgical removal of the apex of the ventricle—approximately 20% of the ventricle's volume—the missing tissue is fully regenerated within 2 months. As a result, the zebrafish has become a favorite model for studying cardiac regeneration, as this vertebrate system combines substantial regenerative capacity with the ability to carry out genetic analyses. Elucidating the mechanisms underlying regeneration should enable us to better understand why the mammalian heart exhibits very limited regenerative capacity despite the presence of cardiac progenitor cells in mouse, rat, and human postnatal myocardium (reviewed in Srivastava and Ivey, 2006Srivastava D. Ivey K.N. Nature. 2006; 441: 1097-1099Crossref PubMed Scopus (124) Google Scholar). By investigating which cells contribute to cardiac regeneration in zebrafish, Lepilina et al., 2006Lepilina A. Coon A.N. Kikuchi K. Holdway J.E. Roberts R.W. Burns C.G. Poss K.D. Cell. 2006; (this issue)Google Scholar further our understanding of cardiac regeneration in vertebrates. Classical regeneration consists of three main steps: (1) dedifferentiation of cells neighboring the site of injury, (2) proliferation of the dedifferentiated (multipotent) cells localized in an area called the blastema, and (3) redifferentiation of the multipotent cells into the terminally differentiated cell types that were lost. However, it has remained unclear whether heart regeneration follows the same sequence of events. In particular, the origin of the new cardiomyocytes that repopulate the site of cardiac injury is not clear. Do they derive exclusively from proliferation of fully differentiated cardiomyocytes? Do neighboring cardiomyocytes first dedifferentiate and then redifferentiate into additional cardiomyocytes? Or do the new cardiomyocytes result from the differentiation of a pool of latent undifferentiated progenitor cells? Lepilina et al., 2006Lepilina A. Coon A.N. Kikuchi K. Holdway J.E. Roberts R.W. Burns C.G. Poss K.D. Cell. 2006; (this issue)Google Scholar provide the first evidence that the new myocardial cells arise from undifferentiated progenitor cells. To monitor myocardial differentiation, the authors made use of double transgenic zebrafish in which a green fluorescent protein (EGFP) and a red fluorescent protein (RFP) are expressed under the control of the cardiac myosin light chain 2 (cmlc2) promoter (that is turned on during myocardial differentiation). EGFP and RFP have different folding properties and stability: EGFP folds faster, but RFP is more stable. Therefore, new cardiomyocytes that arise from undifferentiated progenitor cells would be EGFP+RFP−, whereas those that result from dedifferentiation of existing cardiomyocytes may be EGFP−RFP+. Seven days after surgical resection of the ventricle (termed days postamputation, or dpa), a front of EGFP+RFP− cardiomyocytes was detected at the apical edge of the regenerating tissue, suggesting that the regenerated cardiomyocytes arise from undifferentiated progenitor cells that do not express cmlc2 (Figure 1). Expression of early myocardial markers (nkx2.5, hand2, and tbx20) in cells at the apical edge of the regenerating tissue, starting from 3–4 dpa, suggests that these cells give rise to the new cardiomyocytes. It will be important to confirm this hypothesis using a lineage-tracing technique. Indeed, in previous studies (Raya et al., 2003Raya A. Koth C.M. Buscher D. Kawakami Y. Itoh T. Raya R.M. Sternik G. Tsai H.J. Rodriguez-Esteban C. Izpisua-Belmonte J.C. Proc. Natl. Acad. Sci. USA. 2003; 100: 11889-11895Crossref PubMed Scopus (245) Google Scholar), the inability to clearly detect upregulation of early myocardial markers (nkx2.5, tbx5 and cardiac ankyrin repeat protein, or CARP) in the regenerating heart led to the proposal that the new cardiomyocytes in the regenerating region were unlikely to have derived from an undifferentiated cell population. Lepilina et al., 2006Lepilina A. Coon A.N. Kikuchi K. Holdway J.E. Roberts R.W. Burns C.G. Poss K.D. Cell. 2006; (this issue)Google Scholar did not detect any EGFP−RFP+ cells (indicative of inactivation of cmlc2 expression) at 3 and 7 dpa. These findings suggest that although new cardiomyocytes appear to arise from an undifferentiated progenitor pool, this progenitor pool does not seem to derive from dedifferentiating cardiomyocytes neighboring the site of injury. This model implies that heart regeneration occurs in a different way than classic regeneration, where dedifferentiation is a critical first step. Nevertheless, because rapid dedifferentiation may not be detected by the approach of Lepilina et al., 2006Lepilina A. Coon A.N. Kikuchi K. Holdway J.E. Roberts R.W. Burns C.G. Poss K.D. Cell. 2006; (this issue)Google Scholar, the authors leave room for alternative interpretations. Previous studies (Poss et al., 2002Poss K.D. Wilson L.G. Keating M.T. Science. 2002; 298: 2188-2190Crossref PubMed Scopus (1236) Google Scholar, Raya et al., 2003Raya A. Koth C.M. Buscher D. Kawakami Y. Itoh T. Raya R.M. Sternik G. Tsai H.J. Rodriguez-Esteban C. Izpisua-Belmonte J.C. Proc. Natl. Acad. Sci. USA. 2003; 100: 11889-11895Crossref PubMed Scopus (245) Google Scholar) report that a significant amount of myocardial regeneration results from a high cardiomyocyte-proliferation rate, starting at 7 dpa with a peak at 14 dpa. Lepilina et al., 2006Lepilina A. Coon A.N. Kikuchi K. Holdway J.E. Roberts R.W. Burns C.G. Poss K.D. Cell. 2006; (this issue)Google Scholar used the same technique, that is, incorporation of bromodeoxyuridine (BrdU)—a chemical compound incorporated into cells undergoing DNA synthesis—to label proliferating cells. This analysis revealed that at 7 dpa it is the freshly differentiated cardiomyocyte population (EGFP+RFP−) that is proliferating. It will also be interesting to investigate the proliferation of the myocardial precursors (the nkx2.5-expressing cells) detected at earlier stages in the regeneration process. In other regenerating systems, such as the fish fin, surrounding epidermal tissues play a catalytic role during regeneration (Poss et al., 2000Poss K.D. Shen J. Nechiporuk A. McMahon G. Thisse B. Thisse C. Keating M.T. Dev. Biol. 2000; 222: 347-358Crossref Scopus (249) Google Scholar). Inspired by these findings, Lepilina et al., 2006Lepilina A. Coon A.N. Kikuchi K. Holdway J.E. Roberts R.W. Burns C.G. Poss K.D. Cell. 2006; (this issue)Google Scholar next investigated whether the epicardium—the tissue enveloping the myocardium—plays a role during myocardial regeneration in the zebrafish. The authors observed that by 1–2 dpa the entire epicardial layer (even that surrounding the atrium and outflow tract) expressed markers of embryonic epicardium (raldh2 and tbx18) and began to proliferate. By 7 dpa, the proliferating epicardial cells were restricted to the site of injury. At 14 dpa and as late as 30 dpa, the epicardial cells appeared to invade the regenerating myocardium. Lepilina et al., 2006Lepilina A. Coon A.N. Kikuchi K. Holdway J.E. Roberts R.W. Burns C.G. Poss K.D. Cell. 2006; (this issue)Google Scholar suspected that the invading epicardial cells could play a role in establishing new blood vessels in the regenerating tissue given that this type of invasion occurs during development of the coronary vasculature (Mikawa and Gourdie, 1996Mikawa T. Gourdie R.G. Dev. Biol. 1996; 174: 221-232Crossref PubMed Scopus (495) Google Scholar, Dettman et al., 1998Dettman R.W. Denetclaw Jr., W. Ordahl C.P. Bristow J. Dev. Biol. 1998; 193: 169-181Crossref PubMed Scopus (435) Google Scholar). Therefore, the authors analyzed vascular changes during the regenerative process. They observed that indeed the expression of epicardial markers in the regenerating tissue temporally coincided with the vascularization of new myocardial tissue. Again, it will be important to follow these initial studies with lineage analyses to test the hypothesis that the epicardium gives rise to the endothelial cells within the regenerating tissue. What is the signal that directs epicardial cells to invade and presumably vascularize the regenerating myocardium? Predicting that fibroblast growth factor (Fgf) signaling could promote the invasion process—as this pathway is known to regulate epicardial epithelial-to-mesenchymal transition (EMT) in cultured cells (Morabito et al., 2001Morabito C.J. Dettman R.W. Kattan J. Collier J.M. Bristow J. Dev. Biol. 2001; 234: 204-215Crossref Scopus (152) Google Scholar)—they examined the expression of multiple Fgf ligands and receptors at different times during heart regeneration. In situ analyses revealed that fgf17b was upregulated in the regenerating myocardium (mainly at 7 dpa, but also at 14 and 30 dpa). Additionally, two Fgf17b receptor genes, fgfr2 and fgfr4, showed a similar temporal and spatial expression pattern (fgfr4 mostly at 14 dpa) within or adjacent to the regenerating region. Double in situ analyses of fgfr4 and tbx18 indicate that the fgfr-expressing cells are derived from the epicardium, supporting the hypothesis that Fgf17b in the regenerating myocardium signals through Fgfr2 or Fgfr4 to recruit epicardial cells and promote vascularization of the regenerating region. Additional data indicate that Fgf signaling is essential for complete heart regeneration to occur. When Fgf signaling was blocked in transgenic zebrafish expressing heat-inducible dominant-negative Fgfr1 (Lee et al., 2005Lee Y. Grill S. Sanchez A. Murphy-Ryan M. Poss K.D. Development. 2005; 132: 5173-5183Crossref PubMed Scopus (258) Google Scholar), regeneration was incomplete (Figure 1). In the absence of Fgf signaling, epicardial cells expressing tbx18 were present adjacent to the regenerating region but failed to invade this region, and a scar was formed. Thus, Fgf signaling may facilitate myocardial regeneration through the recruitment of epicardial cells into the regenerating tissue, resulting in neovascularization and completion of the regenerative process. Other interesting aspects of heart regeneration remain to be investigated. These questions include how loss of myocardial tissue is sensed and communicated to the rest of the organ, the origin of the undifferentiated progenitor cells and mode of recruitment, the mechanism of inducing expression of signaling pathway components (such as FgF17b in the regenerating myocardium and Fgfr2/4 in the activated epicardial cells), and how the size of the regenerating region is controlled. Understanding the many facets of heart regeneration in zebrafish ultimately may help in the design of new cardiac regeneration therapies for treating human heart disease. A Dynamic Epicardial Injury Response Supports Progenitor Cell Activity during Zebrafish Heart RegenerationLepilina et al.CellNovember 03, 2006In BriefZebrafish possess a unique yet poorly understood capacity for cardiac regeneration. Here, we show that regeneration proceeds through two coordinated stages following resection of the ventricular apex. First a blastema is formed, comprised of progenitor cells that express precardiac markers, undergo differentiation, and proliferate. Second, epicardial tissue surrounding both cardiac chambers induces developmental markers and rapidly expands, creating a new epithelial cover for the exposed myocardium. Full-Text PDF Open Archive