Figure 7. Schematic overview of morphological and molecular changes upon myocardial ischemia. doi:10.1371/journal.pone.0044692.g007 area from remote sites, as in the healthy heart Wt1-expressing cells have been identified in stem cells niches in the atrioventricular sulcus and apex of the ventricle and, secondly, that Wt1 has been shown to be important for the formation of progenitor cells in the epicardium during development [39]. After the induction of the ischemia the gap between the epicardium and infarcted myocardium initially widens forming an extensive mesenchymal layer. The mesenchymal cells within this layer are virtually all derived from the Wt1-lineage, as they express Wt1, Cre and/or bGal. At this point it is of relevance to note that the Wt1 and Cre are not expressed in this region of the healthy controls, further underscoring the reactivation of the epicardium in the infarcted region. Subsequently, at one and three months post-MI, the mesenchymal layer starts to thin, as the mesenchymal cells start to occupy the space of dead cardiomyocytes. Interesting, in the forming infarct scar more than one third of the cells are positive for bGal, indicating that not only cells of the Wt1-lineage contribute to the scar tissue. This finding is in line with analyses showing a contribution of endocardium-derived cells [35,36], bone Endogenous Epicardial Regenerative Response marrow-derived cells [37], as well as a small contribution of cardiomyocytes [48] to the scar. During development epicardium-derived cells contribute to the intermyocardial fibroblast population and the coronary vessels [39]. To evaluate whether after an infarction the epicardium-derived cells, i.e. the Wt1-lineage positive cells, also contribute to these populations a triple immunofluorescent analysis was performed. This analysis showed that a small portion of the Wt1-lineage positive cells also expressed CD31, qualifying them as endothelial cells. The identification of Wt1-lineage positive coronary endothelial cells was unexpected based on a mouse study [49], but expected based on studies in chicken [50,51]. Our Wt1-lineage analysis together with the analysis of the pattern of expression of Wt1 mRNA and protein suggests that the coronary endothelial cells are de novo formed. Alternatively, the CD31-expressing Wt1-lineage positive cells are the result of outgrowth of coronaries from the border zone into the infarcted area. Although we were not able to identify Wt1 mRNA or protein expression in the border zone of the infarct, it has been reported that after an infarct Wt1 expression is induced in coronary endothelium in the border zone [33]. The identification of a small number of Wt1-lineage positive cardiomyocytes from one month post-MI onward was unexpected. Epicardial-to-myocardial differentiation is a contentious topic based on mouse and chicken studies [18,31,32,34,52]. Our first concern was the specificity of our staining. However, the spatio- temporal pattern of the cardiomyocytes and the fact that they are positive for different markers does underscore the specificity of the staining. Another issue is the fact that only a very limited number of cardiomyocytes is found. Because the labeling of cells in the Cre/lox system is the result of a stochastic process, low numbers of positive cells might be the result of random recombination and thus as such illegitimate labeling. However, this does not seem to be an issue as Wt1-lineage positive myocytes are only observed in the infarct at one and three months post-MI and never in control hearts or in infarcts prior to one month post-MI. Moreover, if the labeling was due to illegitimate recombination then one would expect a random distribution throughout the infarct or even the entire heart, which is not the case. Epicardium-derived cardiomyocytes have been reported in zebra fish upon amputation of the apex [10] and, in a recent study in mice, after a myocardial infarction [18]. Nevertheless, the origin of these cardiomyocytes is largely unknown. Although our Wt1-lineage analysis suggests that the myocytes are direct daughters of the de novo formed epicardial layer covering the infarcted zone, other options are possible. These myocytes might be derived from the stem cell population that has expressed Wt1 early during its life-span and resides in the heart. Alternatively, these cardiomyocytes might be the result of fusion of lineage positive mesenchymal cells with existing cardiomyocytes, a phenomenon also occurring during stem cell transplantation [53]. The observation that most of the myocytes do not have the characteristic adult cardiomyocyte shape and contain only one nucleus, renders this latter option unlikely. Nevertheless, experi- ments need to be performed to falsify this option. Taken together, these observations point to a Wt1-derived population of cells, that is capable of differentiating into the myocardial lineage after a myocardial infarction. When the efficiency of this process could be enhanced, this population forms an attractive source of new cardiomyocytes in a regenerative approach [6]. Pro-fibrotic mesenchymal signaling inhibits cardiomyocyte formation The limited myocardial regenerative capacity of the mouse heart might be hard wired or the net outcome of the integration of PLOS ONE | www.plosone.org 12 September 2012 | Volume 7 | Issue 9 | e44692