Cardiac Regeneration from Activated Epicardium Bram van Wijk, Quinn D. Gunst, Antoon F. M. Moorman, Maurice J. B. van den Hoff* Heart Failure Research Center, Academic Medical Center, Amsterdam, The Netherlands Abstract In contrast to lower vertebrates, the mammalian heart has a very limited regenerative capacity. Cardiomyocytes, lost after ischemia, are replaced by fibroblasts. Although the human heart is able to form new cardiomyocytes throughout its lifespan, the efficiency of this phenomenon is not enough to substitute sufficient myocardial mass after an infarction. In contrast, zebrafish hearts regenerate through epicardial activation and initiation of myocardial proliferation. With this study we obtain insights into the activation and cellular contribution of the mammalian epicardium in response to ischemia. In a mouse myocardial infarction model we analyzed the spatio-temporal changes in expression of embryonic epicardial, EMT, and stem cell markers and the contribution of cells of the Wt1-lineage to the infarcted area. Though the integrity of the epicardial layer overlaying the infarct is lost immediately after the induction of the ischemia, it was found to be regenerated at three days post infarction. In this regenerated epicardium, the embryonic gene program is transiently re-expressed as well as proliferation. Concomitant with this activation, Wt1-lineage positive subepicardial mesenchyme is formed until two weeks post-infarction. These mesenchymal cells replace the cardiomyocytes lost due to the ischemia and contribute to the fibroblast population, myofibroblasts and coronary endothelium in the infarct, and later also to the cardiomyocyte population. We show that in mice, as in lower vertebrates, an endogenous, epicardium-dependent regenerative response to injury is induced. Although this regenerative response leads to the formation of new cardiomyocytes, their number is insufficient in mice but sufficient in lower vertebrates to replace lost cardiomyocytes. These molecular and cellular analyses provide basic knowledge essential for investigations on the regeneration of the mammalian heart aiming at epicardium- derived cells. Citation: van Wijk B, Gunst QD, Moorman AFM, van den Hoff MJB (2012) Cardiac Regeneration from Activated Epicardium. PLoS ONE 7(9): e44692. doi:10.1371/ journal.pone.0044692 Editor: Cara Gottardi, Northwestern University Feinberg School of Medicine, United States of America Received February 16, 2011; Accepted August 10, 2012; Published September 20, 2012 Copyright: 2012 van Wijk et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The presented work was supported by Netherlands Heart Foundation grant 1996M002 and the European Community’s Sixth Framework Program contract (‘‘HeartRepair’’) grant LSHM-CT-2005-018630. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: m.j.vandenhoff@amc.uva.nl Introduction Cardiac disease leads to the highest levels of morbidity and mortality worldwide. Due to their limited regenerative capacity, cardiomyocytes lost as a result of ischemic damage are replaced by non-contractile fibrotic cells rather than by new cardiomyocytes [1,2]. It has long been recognized that a potential cure, infers replenishment of cardiomyocytes from exogenous or endogenous sources. Application of stem cells has gained a lot of interest but the outcome is, thus far, rather disappointing; homing, survival and integration of newly formed cardiomyocytes are serious hurdles in regenerative cardiac medicine [3,4]. Stimulation of the endogenous regenerative capacity seems an attractive alternative for the stem cell approach [5,6]. In adult zebrafish, cardiac regeneration is found upon amputation of the cardiac apex, whereas in the mammalian heart such regeneration is limited to the first week after birth [7]. During this regenerative process the epicardium was found to play a crucial role [8–10]. Also from a developmental stance the epicardium is an interesting source, as (1) the epicardium is derived from a progenitor pool that also provides cardiomyocytes to the inflow of the heart [11], (2) epicardium-derived cells are essential regulators of cardiac growth [12] and (3) the epicardium contributes cells to the coronaries and interstitium [13]. Although it has been reported that embryonic epicardial genes are re-expressed in response to cardiac injury, little is known of the role of the epicardium in homeostasis and regeneration of the adult mammalian heart [14–17]. Recently, it has been shown that upon a myocardial infarction a limited number of epicardium-derived cells have the capacity to contribute cardiomyocytes to the infarct [18], offering a novel inroad to cardiac regeneration. A prerequisite to developing regenerative strategies involving the epicardium is to understand the response of the epicardium to myocardial injury. To investigate the role of the epicardium in endogenous regeneration, we induced a myocardial infarction (MI) in mice in which the Wilm’s Tumor 1 (Wt1)-lineage is genetically labeled. At various time points post-MI the cellular and molecular responses of the epicardium were analyzed. We observed that epicardial cells overlaying the ischemic area had disappeared one day post-MI. The epicardium bordering the ischemic area (border zone) was found to transiently re-express embryonic epicardial markers genes (Wt1, Tbx18, Raldh) and to initiate proliferation. At three days post-MI a new layer of epicardium with extended extracellular matrix had formed over the infarcted area expressing embryonic epicardial marker genes. In this epicardium, genes important for Epithelial-to-Mesenchy- mal-Transition (EMT) were expressed (Snai1, aSMA) and mesenchyme was observed to populate the subepicardial space. Analysis of the Wt1-lineage showed that the newly-formed epicardium and a large portion of the mesenchyme populating PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e44692