various signaling pathways. During early development the extent of the Wt1-expressing progenitor population at the inflow of the heart is regulated by Wnt signaling [43], and the subsequent differentiation into the epicardial or myocardial lineages by the cooperative interaction of Erk-mediated FGF-signaling and Smad- mediated BMP-signaling [11]. In the developing epicardium BMP-mediated myocardial differentiation is inhibited by Erk- mediated phosphorylation of Smad, rendering it incapable of accumulating in the nucleus and conveying the BMP signal [11]. With further development, the epicardium secretes growth factors, like FGF and BMP that stimulate proliferation of the underlying myocardium [30]. Also the adult epicardium was found to secrete growth factors that modulate the extent of the injury after a myocardial infarction [32,42]. Immunofluorescent analysis of the infarcted area identified abundant P-Erk expression in the mesenchyme, as well as very limited and low levels of expression of P-Smad. These data suggest that in the infarcted region, like at the inflow of the developing heart, Erk-mediated FGF-signaling stimulates the formation of non-myocardial cells and inhibits cardiomyocyte differentiation via a cooperative interaction [54]. This idea is further supported by the observation that Fstl1, a natural BMP inhibitor [55–57] is expressed at high levels in the infarct, and as such inhibits cardiomyocyte differentiation. At first glance contradictory to our idea, ectopic expression of Fstl1 [58,59] or inhibition of FGF-signaling [60] reduces infarct size. This reduction in infarct size is, however, not due to formation of new cardiomyocytes, but the result of induced/enhanced revas- cularization, enhancing survival of cardiomyocytes. Also the References 1. Bergmann O, Bhardwaj RD, Bernard S, Zdunek S, Barnabe-Heider F, et al. (2009) Evidence for cardiomyocyte renewal in humans. Science 324: 98–102. 2. Kajstura J, Urbanek K, Perl S, Hosoda T, Zheng H, et al. (2010) Cardiomyogenesis in the adult human heart. Circ Res 107: 305–315. 3. Passier R, van Laake LW, Mummery CL (2008) Stem-cell-based therapy and lessons from the heart. Nature 453: 322–329. 4. Barnett P, van den Hoff MJ (2011) Cardiac regeneration: different cells same goal. Med Biol Eng Comput 49: 723–732. 5. van Wijk B, van den Hoff MJ (2010) Epicardium and myocardium originate from a common cardiogenic precursor pool. Trends Cardiovasc Med 20: 1–7. 6. Kuhn EN, Wu SM (2010) Origin of cardiac progenitor cells in the developing and postnatal heart. J Cell Physiol 225: 321–325. 7. Porrello ER, Mahmoud AI, Simpson E, Hill JA, Richardson JA, et al. (2011) Transient regenerative potential of the neonatal mouse heart. Science 331: 1078–1080. 8. Jopling C, Sleep E, Raya M, Marti M, Raya A, et al. (2010) Zebrafish heart regeneration occurs by cardiomyocyte dedifferentiation and proliferation. Nature 464: 606–609. 9. Kikuchi K, Holdway JE, Werdich AA, Anderson RM, Fang Y, et al. (2010) Primary contribution to zebrafish heart regeneration by gata4(+) cardiomyo- cytes. Nature 464: 601–605. 10. Lepilina A, Coon AN, Kikuchi K, Holdway JE, Roberts RW, et al. (2006) A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration. Cell 127: 607–619. 11. van Wijk B, van den Berg G, Abu-Issa R, Barnett P, van der Velden S, et al. (2009) Epicardium and myocardium separate from a common precursor pool by crosstalk between bone morphogenetic protein- and fibroblast growth factor- signaling pathways. Circ Res 105: 431–441. 12. Ieda M, Tsuchihashi T, Ivey KN, Ross RS, Hong TT, et al. (2009) Cardiac fibroblasts regulate myocardial proliferation through beta1 integrin signaling. Dev Cell 16: 233–244. 13. Munoz-Chapuli R, Macias D, Gonzalez-Iriarte M, Carmona R, Atencia G, et al. (2002) The epicardium and epicardial-derived cells: multiple functions in cardiac development. Rev Esp Cardiol 55: 1070–1082. 14. Di Meglio F, Castaldo C, Nurzynska D, Romano V, Miraglia R, et al. (2010) Epithelial-mesenchymal transition of epicardial mesothelium is a source of cardiac CD117-positive stem cells in adult human heart. J Mol Cell Cardiol 49: 719–727. 15. Limana F, Bertolami C, Mangoni A, Di CA, Avitabile D, et al. (2009) Myocardial infarction induces embryonic reprogramming of epicardial c-kit(+) cells: Role of the pericardial fluid. J Mol Cell Cardiol 48: 609–618. 16. Limana F, Zacheo A, Mocini D, Mangoni A, Borsellino G, et al. (2007) Identification of myocardial and vascular precursor cells in human and mouse epicardium. Circ Res 101: 1255–1265. Endogenous Epicardial Regenerative Response matricellular protein, Periostin, which directs differentiation of mesenchymal cells into fibroblasts [61,62] and upon disruption results in reduced infarct size [63], was found to be expressed at high levels in the infarct. General conclusion Taken together, this analysis shows that a regenerative response, similar to the response observed in fish, is initiated in the mouse upon MI, suggesting that an evolutionary conserved mechanism persists in the mammalian heart. However, unlike in fish, the regenerative response in mammalian heart is abrogated, resulting in the formation of only a limited number of new cardiomyocytes. Our findings provide basic knowledge that will be useful for the development of new strategies to enhance the endogenous regenerative capacity of the epicardial lineage. Acknowledgments We thank Ben Middellaar for the technical support on the LAD-ligation model, John Burch for the Wt1Cre mouse line, and Drs Phil Barnett and Jan Ruijter for critically reviewing the manuscript. Author Contributions Conceived and designed the experiments: BVW MVH AFM. Performed the experiments: BVW QDG MVH. Analyzed the data: BVW QDG MVH. Contributed reagents/materials/analysis tools: BVW QDG MVH. Wrote the paper: BVW QDG MVH AFM. 17. Di Nardo P, Forte G, Ahluwalia A, Minieri M (2010) Cardiac progenitor cells: Potency and control. J Cell Physiol 224: 590–600. 18. Smart N, Bollini S, Dube KN, Vieira JM, Zhou B, et al. (2011) De novo cardiomyocytes from within the activated adult heart after injury. Nature 474: 640–644. 19. Wessels A, van den Hoff MJ, Adamo RF, Phelps AL, Lockhart MM, et al. (2012) Epicardially derived fibroblasts preferentially contribute to the parietal leaflets of the atrioventricular valves in the murine heart. Dev Biol 366: 111–124.. 20. Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21: 70–71. 21. Timmers L, Sluijter JP, van Keulen JK, Hoefer IE, Nederhoff MG, et al. (2008) Toll-like receptor 4 mediates maladaptive left ventricular remodeling and impairs cardiac function after myocardial infarction. Circ Res 102: 257–264. 22. Ruijter JM, Ramakers C, Hoogaars WM, Karlen Y, Bakker O, et al (2009) Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res 37: e45. 23. Somi S, Klein ATJ, Houweling AC, Ruijter JM, Buffing AAM, et al. (2006) Atrial and ventricular myosin heavy-chain expression in the developing chicken heart: strengths and limitations of non-radioactive in situ hybridization. J Histochem Cytochem 54: 649–664. 24. Snarr BS, O’Neal JL, Chintalapudi MR, Wirrig EE, Phelps AL, et al. (2007) Isl1 Expression at the Venous Pole Identifies a Novel Role for the Second Heart Field in Cardiac Development. Circ Res 101: 971–974. 25. Kraus F, Haenig B, Kispert A (2001) Cloning and expression analysis of the mouse T-box gene Tbx18. Mech Dev 100: 83–86. 26. Haenig B, Kispert A (2004) Analysis of TBX18 expression in chick embryos. Dev Genes Evol 214: 407–411. 27. Moss JB, Xavier-Neto J, Shapiro MD, Nayeem SM, McCaffery P, et al. (1998) Dynamic patterns of retinoic acid synthesis and response in the developing mammalian heart. Dev Biol 199: 55–71. 28. Carmona R, Gonzalez-Iriarte M, Perez-Pomares JM, Munoz-Chapuli R (2001) Localization of the Wilm’s tumour protein WT1 in avian embryos. Cell Tissue Res 303: 173–186. 29. Armstrong JF, Pritchard-Jones K, Bickmore WA, Hastie ND, Bard JB (1993) The expression of the Wilms’ tumour gene, WT1, in the developing mammalian embryo. Mech Dev 40: 85–97. 30. Lavine KJ, Ornitz DM (2007) Rebuilding the coronary vasculature: hedgehog as a new candidate for pharmacologic revascularization. Trends Cardiovasc Med 17: 77–83. 31. Zhou B, Ma Q, Rajagopal S, Wu SM, Domian I, et al. (2008) Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart. Nature 454: 109–113. PLOS ONE | www.plosone.org 13 September 2012 | Volume 7 | Issue 9 | e44692