Endogenous Epicardial Regenerative Response infarcted area (G) and the infarct proper (J), but not covering remote, healthy myocardium (K, M & O). One week post-MI (P–U), the pattern of expression of Wt1 (R, S) and Cre (T, U) is essentially the same as at three days post-MI. Three months post-MI (U–W), Wt1 (W) and Cre (X) are, like in the control heart, not longer detectable. Abbreviations: LV: left ventricle, RV: right ventricle, Epi: epicardium. The bar is 100 mm. doi:10.1371/journal.pone.0044692.g002 Since this Wt1Cre mouse line encodes a constitutive, rather than an inducible, Cre, the lineage analysis will not only show the effects that are the result of the induced myocardial infarction, but also the developmental history. To visualize the result of the developmental expression of Wt1 in the adult heart, we crossed male Wt1Cre with female R26R reporter mice and used immunofluorescent staining to identify the ß-Galactosidase (ß- Gal) expressing cells. As expected. the entire epicardium and adjacent mesenchyme, a subset of the endothelial cells of the coronaries and intermyocardial fibroblasts were found to express ß-Gal in the adult hearts (Figure 3A,B). We did not identify ß-Gal- expressing cardiomyocytes in the ventricular free wall, which is in line with our developmental analysis [19] but in contrast to the Wt1 Cre knock-in lines [31,34]. Next we established the ß-Gal-expression pattern at various time points after permanent ligation of the LAD. To identify the ischemic area, we co-stained the sections for cTnI. The cTnI expression level is decreased at 1 day post-MI and below detection from three days post-MI onward in the infarcted region (Figure 3). At 1 day post-MI the ß-Gal-expressing epicardium overlying the border zone of the infarcted area showed interruptions, and was largely absent from the infarcted area (Figure 3C–D). Classic histological staining of neighboring sections confirmed that the epicardial layer covering the ischemic area had disintegrated (data not shown), which is in line with previously published data [14]. Three days post-MI the infarcted area was found to be covered with a contiguous ß-Gal-expressing epicardial layer (figure 3E–H). These observations suggest that the epicardial layer overlaying the ischemic area is lost and regenerated within three days after the induction of ischemia. At three days post MI not only the epicardium was regenerated but also the space between the myocardium and epicardium covering the border zone of the infarction had widened and had become populated with ß-Gal- expressing mesenchymal cells (Figure 2F). In areas remote of the infarction and in controls, there was virtually no space between the epicardium and the ventricular myocardium (compare Figure 3F, J with 3H, K). At one week post-MI the space between the ß-Gal- positive epicardium and the myocardium had further widened and was no longer restricted to the border zone but included the entire infarct and was extensively populated with ß-Gal-expressing mesenchymal cells (Figure 3I–K). At one month post-MI, the wide ß-Gal-positive mesenchymal layer was still present in the border zone, but had disappeared from the infarcted area. ß-Gal- positive cells were now found throughout the infarcted area, suggesting that the subepicardial mesenchyme had invaded and replaced dead cardiomyocytes in the infarct (Figure 3L). This became even more evident at three months post-MI (data not shown). Although ß-Gal-positive cells contribute to the infarcted region not all cells are positive, increasing from 1462% of the cells at one week post-MI to 3865% of the non-myocardial cells within the infacted area, excluding the epicardium and subepicardial mesenchyme. These findings indicate that the non-myocytes in the infacted area are derived from other ancestries as well, being the endocardium [35,36] and the bone marrow [37]. To gain insight into the phenotype of the ß-Gal-positive cells in the infarcted region triple immunofluorescent staining was performed. Based on this analysis the ß-Gal-expressing cells could be subdivided in four groups: (i) Cells co-expressing a-Smooth Muscle Actin (a-SMA) (Figure 3M), that were found throughout the infarcted area and adjacent to blood vessels. (ii) Cells co- expressing PECAM (CD31) that were located within coronary vessels (Figure 3N). (iii) Cells co-expressing cTnI, qualifying them as cardiomyocytes (Figure 3O–P). (iv) Cells co-expressing none of the three above markers, qualifying them as interstitial cells (Figure 3M–P). It should be noted that the cardiomyocytes comprise small islands of small rounded cells that most often do not show the typical architecture of adult cardiomyocytes. Due to their non-random distribution it is impossible to reliably quantify them, nevertheless we estimate that this population comprises a few hundred cells per infarct. With respect to this observation it is relevant to note that cTnI and ß-Gal double-positive cells (1) were only found at 1 and 3 months post-MI, (2) were never found in the left ventricular free wall outside the infarcted area, and (3) were never found in the ventricular free wall of controls. (4) Moreover, these cells were besides positive for cTnI also positive for other cardiomyocytes markers, like sarcoplasmic reticulum calcium ATPase 2a (SERCA2a). Proliferation of Epicardial and mesenchymal cells The observation that the epicardium overlaying the infarcted region is regenerated within the first three days after the infarct and the formation of a thick layer of subepicardial mesenchyme over the infarcted area, suggests that the healthy epicardium from the border zone grows over the infarct and that the newly formed epicardium proliferates and undergoes EMT to provide the subepicardial mesenchyme. To investigate these possibilities, mice were labeled with BrdU for 4 hours before sacrifice. As a positive control gut tissue was isolated and analyzed in parallel (Figure 4D). In control hearts, as expected [38], only an incidental non- myocardial cells was found to have incorporated BrdU (Figure 4A– C). One day post-MI, BrdU-incorporation was observed in the epicardial cells overlying the border zone of the infarct (Figure 4E) and in a low number of epicardial cells covering the atria (data not shown). We also observed BrdU incorporation in endocardial cells throughout the entire heart. During the following two weeks post- MI, BrdU incorporation was detected in the epicardium and mesenchymal cells overlying the infarct, but not in epicardial cells covering uninjured remote ventricular myocardium (Figure 4F–L). At 1 and 3 months post-MI, BrdU-incorporation was largely similar to controls, except for an occasional individual mesenchy- mal cell in the border zone of the infarct (Figure 4M–P). These observations suggest that the lost epicardium is regenerated from healthy epicardium covering the border zone. Secondly, the finding that the epicardium overlaying the infarct and its adjacent mesenchyme is not only ß-Gal-positive but also has incorporated BrdU, further underscores the idea that the subepicardial mesenchyme is de novo formed by EMT from the overlaying epicardium. Subepicardial mesenchyme formation by EMT Both the proliferation and lineage analysis point to de novo mesenchyme formation from the overlaying epicardium by EMT. To further substantiate this idea the pattern of expression of Snai1 and a-SMA, markers of EMT during embryogenesis [39], were determined in the region of the infarct. Concomitant with the appearance of mesenchymal cells adjacent to the epicardium covering the border zone and the infarcted area, expression of Snai1 (Figure 5A–C) and a-SMA (Figure 5J–M) were found in PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e44692