Endogenous Epicardial Regenerative Response Figure 5. Post-ischemic mesenchyme formation. Snai1 mRNA is present from three days onwards in the subepicardial mesenchyme of the apex and LV covering the infarct (A), and border zone (B). qPCR analysis (C) showed that Snai1 is already induced at one day post-MI and remains high up to two weeks post-MI, after which it gradually returns to control levels. Its close relative Snai2 shows a similar activation pattern as Snai1, with the exception that Snai2 is already detected in the healthy control heart. Within the border zone (D) and infarcted area (E) the matricellular proteins Follistatin-like1 (E,H) and Periostin (F,I) are expressed at high levels in the non-myocardial cells (cf D,G). In control hearts, a-SMA is present in the smooth muscle layer of the coronaries (J). At one and two weeks post-MI a-SMA is detected in the subepicardial mesenchyme and in mesenchymal cells located in the myocardium adjacent to the epicardium overlaying the infarct (K,L). At one and two weeks post-MI a-SMA-postive subepicardial mesenchyme is also found in the atria (M). P-Smad is only found in a small minority of subepicardial mesenchymal cells (arrow) directly flanking myocardial cells and adjacent to the infarct (N). Whereas P-Erk is detected in the nucleus of a subset of epicardial cells in controls and after an infarction, as well as in the cytoplasm and nucleus of an extensive subset of the subepicardial mesenchyme (O–Q). The bar is 100 mm. doi:10.1371/journal.pone.0044692.g005 family, Follistatin-like-1 (Figure 5E,H) and Periostin (Figure 5F,I), that both have been shown to be involved in mesenchyme differentiation in the developing heart. Fstl1 and Periostin mRNA were both present in the infarcted zone, with Fstl1 being more extensively expressed than Periostin (Figure 5D–I). Both mRNAs were most abundantly expressed at one week post-MI, after which their expression gradually decreased to very low, but still detectable levels, at two weeks and becoming undetectable from one month post-MI onward. Regulators of mesenchyme differentiation Supplementing medium conditioned by EPDCs to mice, was found to reduce the size of the infarct and improve cardiac function [32]. Recently, expression of Wnt1 was found to be induced in the epicardium and adjacent infarct tissue, eliciting a pro-fibrotic response [42]. During development a progenitor cell population located upstream of the inflow of the heart contributes cells to the myocardium and epicardium. Inhibiting Wnt signaling during development by applying recombinant Wif1 to the developing embryo was found to increase this progenitor pool [43]. The cooperative interaction of Smad-mediated BMP and Erk-mediated FGF-signaling subsequently directs the differentia- tion of these progenitors into either the myocardial or epicardial lineages [11,44]. Since the lineage analysis had identified a small population of ß-Gal-positive cardiomyocytes, we determined whether P-Smad and P-Erk are expressed after a myocardial infarction. P-Smad was found to be expressed at low levels in mesenchymal cells flanking the cardiomyocytes of the border zone and in scattered cells throughout the infarcted area (Figure 5N). P- Erk, on the other hand, was observed in a small portion of epicardial cells in control hearts. At three days and one week post- MI P-Erk was observed at high levels in the mesenchyme adjacent to the epicardium overlaying the infarcted area (Figure 5O–Q). At one month post-MI, the mesenchymal cells expressing P-Erk were only found in mesenchymal cells in the border zone of the infarct. At three months post-MI the expression pattern was found to be similar to controls. P-Smad was found to be expressed at low levels in mesenchymal cells flanking the cardiomyocytes of the border zone and in scattered cells throughout the infarcted area (Figure 5N). Taken together the pattern of expression of P-Erk suggests a role in epicardial EMT, and that of P-Smad a role in the differentiation of cardiomyocytes in the infarcted. Expression of stem cell markers in adult healthy and infarcted hearts Upon amputation of the cardiac apex in zebrafish, the epicardium is activated, and myocardial cells start to proliferate [9,10,45]. Like the zebrafish heart, the neonatal mouse heart is able to respond to injury [7], whereas in the adult mouse heart proliferation of cardiomyocytes after a myocardial infarction was not observed (Figure 4) [38]. Also in this study we did not observe any BrdU incorporation in adult cardiomyocytes, suggesting that it is very unlikely that the bGal-expressing cardiomyocytes are derived from existing cardiomyocytes by proliferation. As an alternative to de novo differentiation of epicardium-derived cells, the cardiomyocytes might originate from differentiation of stem cells that express or have expressed Wt1. The latter option seems to be a likely option because Wt1 has been shown to be important for the formation of progenitor cells in the epicardium during development [39], and, secondly, the adult epicardium has been suggested to be a source of stem cells [46]. To evaluate the changes in the potential progenitor population, we determined the pattern of expression of Platelet Derived Growth Factor a (PDGFRa), CD34 and cKit by immunofluorescence (Figure 6). Unfortunately, it is technically impossible to perform reliable triple immunu- fluorescent staining for ß-Gal and these stem cell markers because the primary antibodies are raised in the same species. In the healthy heart PDGFRa, CD34 and cKit were detected in the epicardium covering the atria, atrioventricular sulcus and apex. PDGFRa and CD34 were also detected in a subset of mesenchymal cells in the atrioventricular sulcus (Figure 6A, D, I). This finding is in line with the identified stem cell niches [47] and with the sites of expression of Wt1. At three days post-MI, cKit, but not PDGFRa and CD34, expression was observed in a portion of the epicardial cells that cover the border zone of the infarct (Figure 6E). At one and two weeks post-MI expression of all three markers was found in a subset of the mesenchymal cells adjacent to the epicardium overlaying the infarct (Figure 6B, C, F– H, K–L). From one month onward the expression of the stem cell markers was no longer detected in the area of the infarct anymore, but only in the regions found in controls (data not shown). Since the spatio-temporal pattern of expression of the stem cell markers and the cardiomyocytes markers do not overlap in the region of the infarct, this analysis does not imply a direct association. Since our analysis has shown that a large part, if not all, subepicardial cells overlaying the infarct are formed de novo by EMT from overlaying epicardium, one might speculate that after an infarction the epicardium-derived stem cell population expands and provides the precursors of the various Wt1-derived populations (Figure 3 M–P). Nevertheless, the origin of the Wt1-lineage positive cardiomyocytes remains enigmatic. Discussion Traditionally, the epicardium has been regarded as a mesothe- lial sheath protecting the underlying myocardium and producing the pericardial fluid facilitating cardiac contraction. However, recent research has revealed that the epicardium is far from being a static anatomical structure, but plays a pivotal role in homeostasis and regeneration of the adult heart. Using the epicardium as a source of cells in novel regenerative strategies might, therefore, be an attractive option. A prerequisite for developing such novel strategies is the understanding of the cellular and molecular changes in the epicardium during regeneration. PLOS ONE | www.plosone.org 10 September 2012 | Volume 7 | Issue 9 | e44692