Alexander von Gise

Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart

The heart is formed from cardiogenic progenitors expressing the transcription factors Nkx2-5 and Isl1 (refs 1 and 2). These multipotent progenitors give rise to cardiomyocyte, smooth muscle and endothelial cells, the major lineages of the mature heart. Here we identify a novel cardiogenic precursor marked by expression of the transcription factor Wt1 and located within the epicardium—an epithelial sheet overlying the heart. During normal murine heart development, a subset of these Wt1+ precursors differentiated into fully functional cardiomyocytes. Wt1+ proepicardial cells arose from progenitors that express Nkx2-5 and Isl1, suggesting that they share a developmental origin with multipotent Nkx2-5+ and Isl1+ progenitors. These results identify Wt1+ epicardial cells as previously unrecognized cardiomyocyte progenitors, and lay the foundation for future efforts to harness the cardiogenic potential of these progenitors for cardiac regeneration and repair.

Adult mouse epicardium modulates myocardial injury by secreting paracrine factors

The epicardium makes essential cellular and paracrine contributions to the growth of the fetal myocardium and the formation of the coronary vasculature. However, whether the epicardium has similar roles postnatally in the normal and injured heart remains enigmatic. Here, we have investigated this question using genetic fate-mapping approaches in mice. In uninjured postnatal heart, epicardial cells were quiescent. Myocardial infarction increased epicardial cell proliferation and stimulated formation of epicardium-derived cells (EPDCs), which remained in a thickened layer on the surface of the heart. EPDCs did not adopt cardiomyocyte or coronary EC fates, but rather differentiated into mesenchymal cells expressing fibroblast and smooth muscle cell markers. In vitro and in vivo assays demonstrated that EPDCs secreted paracrine factors that strongly promoted angiogenesis. In a myocardial infarction model, EPDC-conditioned medium reduced infarct size and improved heart function. Our findings indicate that epicardium modulates the cardiac injury response by conditioning the subepicardial environment, potentially offering a new therapeutic strategy for cardiac protection.

Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction

In a cell-free approach to regenerative therapeutics, transient application of paracrine factors in vivo could be used to alter the behavior and fate of progenitor cells to achieve sustained clinical benefits. Here we show that intramyocardial injection of synthetic modified RNA (modRNA) encoding human vascular endothelial growth factor-A (VEGF-A) results in the expansion and directed differentiation of endogenous heart progenitors in a mouse myocardial infarction model. VEGF-A modRNA markedly improved heart function and enhanced long-term survival of recipients. This improvement was in part due to mobilization of epicardial progenitor cells and redirection of their differentiation toward cardiovascular cell types. Direct in vivo comparison with DNA vectors and temporal control with VEGF inhibitors revealed the greatly increased efficacy of pulse-like delivery of VEGF-A. Our results suggest that modRNA is a versatile approach for expressing paracrine factors as cell fate switches to control progenitor cell fate and thereby enhance long-term organ repair.

YAP1, the nuclear target of Hippo signaling, stimulates heart growth through cardiomyocyte proliferation but not hypertrophy

Heart growth is tightly controlled so that the heart reaches a predetermined size. Fetal heart growth occurs through cardiomyocyte proliferation, whereas postnatal heart growth involves primarily physiological cardiomyocyte hypertrophy. The Hippo kinase cascade is an important regulator of organ growth. A major target of this kinase cascade is YAP1, a transcriptional coactivator that is inactivated by Hippo kinase activity. Here, we used both genetic gain and loss of Yap1 function to investigate its role in regulating proliferative and physiologic hypertrophic heart growth. Fetal Yap1 inactivation caused marked, lethal myocardial hypoplasia and decreased cardiomyocyte proliferation, whereas fetal activation of YAP1 stimulated cardiomyocyte proliferation. Enhanced proliferation was particularly dramatic in trabecular cardiomyocytes that normally exit from the cell cycle. Remarkably, YAP1 activation was sufficient to stimulate proliferation of postnatal cardiomyocytes, both in culture and in the intact heart. A dominant negative peptide that blocked YAP1 binding to TEAD transcription factors inhibited YAP1 proliferative activity, indicating that this activity requires YAP1–TEAD interaction. Although Yap1 was a critical regulator of cardiomyocyte proliferation, it did not influence physiological hypertrophic growth of cardiomyocytes, because postnatal Yap1 gain or loss of function did not significantly alter cardiomyocyte size. These studies demonstrate that Yap1 is a crucial regulator of cardiomyocyte proliferation, cardiac morphogenesis, and myocardial trabeculation. Activation of Yap1 in postnatal cardiomyocytes may be a useful strategy to stimulate cardiomyocyte expansion in therapeutic myocardial regeneration.

Endocardial and epicardial epithelial to mesenchymal transitions in heart development and disease

Epithelial to mesenchymal transition (EMT) converts epithelial cells to mobile and developmentally plastic mesenchymal cells. All cells in the heart arise from one or more EMTs. Endocardial and epicardial EMTs produce most of the noncardiomyocyte lineages of the mature heart. Endocardial EMT generates valve progenitor cells and is necessary for formation of the cardiac valves and for complete cardiac septation. Epicardial EMT is required for myocardial growth and coronary vessel formation, and it generates cardiac fibroblasts, vascular smooth muscle cells, a subset of coronary endothelial cells, and possibly a subset of cardiomyocytes. Emerging studies suggest that these developmental mechanisms are redeployed in adult heart valve disease, in cardiac fibrosis, and in myocardial responses to ischemic injury. Redirection and amplification of disease-related EMTs offer potential new therapeutic strategies and approaches for treatment of heart disease. Here, we review the role and molecular regulation of endocardial and epicardial EMT in fetal heart development, and we summarize key literature implicating reactivation of endocardial and epicardial EMT in adult heart disease.

WT1 regulates epicardial epithelial to mesenchymal transition through β-catenin and retinoic acid signaling pathways.

An epithelial sheet, the epicardium, lines the surface of the heart. In the developing embryo, the epicardium expresses the transcriptional regulator Wilms Tumor Gene 1 (Wt1). Through incompletely understood mechanisms, Wt1 inactivation derails normal heart development. We investigated mechanisms by which Wt1 regulates heart development and epicardial epithelial to mesenchymal transition (EMT). We used genetic lineage tracing approaches to track and isolate epicardium and epicardium derivatives in hearts lacking Wt1 (Wt1(KO)). Wt1(KO) hearts had diminished proliferation of compact myocardium and impaired coronary plexus formation. Wt1(KO) epicardium failed to undergo EMT. Wt1(KO) epicardium expressed reduced Lef1 and Ctnnb1 (β-catenin), key components of the canonical Wnt/β-catenin signaling pathway. Wt1(KO) epicardium expressed decreased levels of canonical Wnt downstream targets Axin2, Cyclin D1, and Cyclin D2 and exhibited decreased activity of the Batgal Wnt/β-catenin reporter transgene, suggestive of diminished canonical Wnt signaling. Hearts with epicardium-restricted Ctnnb1 loss of function resembled Wt1(KO) hearts and also failed to undergo epicardial EMT. However, Ctnnb1 inactivation did not alter WT1 expression, positioning Wt1 upstream of canonical Wnt/β-catenin signaling. Wnt5a, a prototypic non-canonical Wnt with enriched epicardial expression, and Raldh2, a key regulator of retinoic acid signaling confined to the epicardium, were also markedly downregulated in Wt1(KO) epicardium. Hearts lacking Wnt5a or Raldh2 shared phenotypic features with Wt1(KO). Although Wt1 has been proposed to regulate EMT by repressing E-cadherin, we detected no change in E-cadherin in Wt1(KO) epicardium. Collectively, our study shows that Wt1 regulates epicardial EMT and heart development through canonical Wnt, non-canonical Wnt, and retinoic acid signaling pathways.

PRC2 directly methylates GATA4 and represses its transcriptional activity

Polycomb-repressive complex 2 (PRC2) promotes tissue-specific differentiation by depositing trimethylated histone H3 Lys 27 (H3K27me3) epigenetic marks to silence ectopic gene expression programs. Here, we show that EZH2, the catalytic subunit of PRC2, is required for cardiac morphogenesis. Both in vitro and in fetal hearts, EZH2 interacted with cardiac transcription factor GATA4 and directly methylated it at Lys 299. PRC2 methylation of GATA4 attenuated its transcriptional activity by reducing its interaction with and acetylation by p300. Our results reveal a new mechanism of PRC2-mediated transcriptional repression in which PRC2 methylates a transcription factor to inhibit its transcriptional activity.

Polycomb Repressive Complex 2 Regulates Normal Development of the Mouse Heart

Rationale: Epigenetic marks are crucial for organogenesis, but their role in heart development is poorly understood. Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27, which establishes H3K27me3 repressive epigenetic marks that promote tissue-specific differentiation by silencing ectopic gene programs. Objective: We studied the function of PRC2 in murine heart development using a tissue-restricted conditional inactivation strategy. Methods and Results: Inactivation of the PRC2 subunit Ezh2 by Nkx2–5Cre (Ezh2NK) caused lethal congenital heart malformations, namely, compact myocardial hypoplasia, hypertrabeculation, and ventricular septal defect. Candidate and genome-wide RNA expression profiling and chromatin immunoprecipitation analyses of Ezh2NK heart identified genes directly repressed by EZH2. Among these were the potent cell cycle inhibitors Ink4a/b (inhibitors of cyclin-dependent kinase 4 A and B), the upregulation of which was associated with decreased cardiomyocyte proliferation in Ezh2NK. EZH2-repressed genes were enriched for transcriptional regulators of noncardiomyocyte expression programs such as Pax6, Isl1, and Six1. EZH2 was also required for proper spatiotemporal regulation of cardiac gene expression, because Hcn4, Mlc2a, and Bmp10 were inappropriately upregulated in ventricular RNA. PRC2 was also required later in heart development, as indicated by cardiomyocyte-restricted TNT-Cre inactivation of the PRC2 subunit Eed. However, Ezh2 inactivation by TNT-Cre did not cause an overt phenotype, likely because of functional redundancy with Ezh1. Thus, early Ezh2 inactivation by Nk2–5Cre caused later disruption of cardiomyocyte gene expression and heart development. Conclusions: Our study reveals a previously undescribed role of EZH2 in regulating heart formation and shows that perturbation of the epigenetic landscape early in cardiogenesis has sustained disruptive effects at later developmental stages.

Nkx2-5- and Isl1-expressing cardiac progenitors contribute to proepicardium

Correct delineation of the hierarchy of cardiac progenitors is a key step to understanding heart development, and will pave the way for future use of cardiac progenitors in the treatment of heart disease. Multipotent Nkx2-5 and Isl1 cardiac progenitors contribute to cardiomyocyte, smooth muscle, and endothelial lineages, which constitute the major lineages of the heart. Recently, progenitors located within the proepicardium and epicardium were reported to differentiate into cardiomyocytes, as well as smooth muscle and endothelial cells. However, the relationship of these proepicardial progenitors to the previously described Nkx2-5 and Isl1 cardiac progenitors is incompletely understood. To address this question, we performed in vivo Cre-loxP-based lineage tracing. Both Nkx2-5- and Isl1-expressing progenitors contributed to the proepicardium and expressed Wt1 and Tbx18, markers of proepicardial progenitor cells. Interestingly, Nkx2-5 knockout resulted in abnormal proepicardial development and decreased expression of Wt1, suggesting a functional role for Nkx2-5 in proepicardium formation. Taken together, these results suggest that Nkx2-5 and/or Isl1 cardiac progenitors contribute to proepicardium during heart development.

Genetic fate mapping demonstrates contribution of epicardium-derived cells to the annulus fibrosis of the mammalian heart.

The annulus fibrosis electrically insulates the atria and ventricles, allowing the timed sequential beating of these structures that is necessary for efficient heart function. Abnormal development of the annulus fibrosis leads to persistence of accessory electrical pathways from atria to ventricles, providing the anatomical substrate for re-entrant cardiac arrhythmias such as Wolff-Parkinson-White syndrome. To better understand the development of the annulus fibrosis and the etiology of these cardiac arrhythmias, we used Cre-LoxP technology to assess the contribution of epicardium derived cells (EPDCs) to the annulus fibrosis. We found that EPDCs migrated into the region of the forming annulus fibrosis, marked by the protein periostin. These EPDCs also stained positive for procollagen I, suggesting that the EPDCs themselves synthesize proteins of the annulus fibrosis. To further test the hypothesis that EPDCs contribute to cells that synthesize the annulus fibrosis, we purified genetically marked EPDCs from the atrioventricular region and measured gene expression by quantitative PCR. These EPDCs were highly enriched for mRNAs encoding periostin, procollagen I, fibronectin I, vimentin, discoidin domain receptor 2, and tenascin C, markers of fibroblasts and components of the annulus fibrosis. In addition, these EPDCs were highly enriched for Snail, Smad1, Slug, and Twist1, markers for epithelial-to-mesenchymal transition (EMT), and a metalloprotease, Mmp2, that contributes to cellular migration. Our work provides for the first time definitive evidence that epicardium contributes to formation of the mammalian annulus fibrosis through EMT. Abnormalities of this differentiation process may underlie development of some forms of re-entrant atrioventricular tachycardia.