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

Abstract

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.

Figure 1. Annulus fibrosis precursors derive from the epicardium

(A–C) Detection of CreERT2 expression in epicardium at E10.5, E13.5, E16.5, using an antibody to the estrogen receptor (ESR1) portion of the CreERT2 fusion protein. Boxes 1, or 2, or 3, are shown at higher magnification. Bar = 100 μm. (D) Schematic depicting the lineage tracing strategy. Tamoxifen (Tam) was injected intraperitoneally at E10.5 to induce Cre recombinase activity. (E) Immunofluorescence staining of periostin (green) and lineage trace marker β-gal (red) at E15.5. A subset of EPDCs forms the periostin+ annulus fibrosis (white arrows). 1, left AV groove; 2, right AV groove. Bar = 100 μm. White arrowheads indicate EPDCs that do not co-express periostin. (F) X-gal (blue; EPDCs) and periostin (red) staining of the consecutive sections. Arrows indicate periostin+ EPDCs in annulus fibrosis. Bar=50 μm. (G) Atrium and ventricular myocardium (desmin, red) were partially separated by EPDCs (blue) in annulus fibrosis (arrows). Bar = 50 μm.

Figure 2. Fatemap of EPDCs in annulus fibrosis using Rosa26^mTmG reporter

(A) Schematic depicting the fate map strategy. Tamoxifen (Tam) was injected at E10.5 to induce Cre recombinase activity. (B) Lineage trace of EPDCs by GFP staining and co-staining with periostin (E15.5) Magnifications of left and right AV grooves are shown in (C) and (D), respectively. Bar = 50 μm.

Figure 3. Fate map of EPDCs in annulus fibrosis at E16.5

Immunostaining of E16.5 Wt1^CreERT2/+;Rosa26^mTmG/+ embro heart annulus region by EPDC lineage trace marker β-gal (red). (A, B) Co-immunostaining of atrioventricular junction of two different embryos by periostin (green) showed co-expression with β-gal (arrows). A subset of EPDCs did not express periostin (white arrowheads). (C) Co-immunostaining of ventricular myocardium by perisotin. Periostin was not robustly detected in epicardium (white arrowheads). (D) Co-immunostaining of heart annulus region with cardiomyocyte marker TNNT2 (green). Epi, epicardium; Endo, endocardium. V, ventricle. Bar = 50 μm.

Figure 4. Fate map of EPDCs in annulus fibrosis at E17.5

(A) At E17.5, EPDCs (β-gal, red) were positive for periostin (green) in annulus fibrosis (white arrows). (B–C) Consecutive sections stained with X-gal and immunostained for periostin or desmin demonstrated EPDCs contribution separation of atrium and ventricles by annulus fibrosis (black arrows). Bar= 50 μm.

Figure 5. EPDCs in the annulus fibrosis express the annulus fibrosis component procollagen I

(A) E15.5 embryo heart was stained with lineage trace marker β-gal (red) and procollagen I (ProColl, green). White arrowheads indicate epicardial expression of pro-collagen I. (B–C) Magnification of left and right AF. Arrows indicate co-expression of β-gal and pro-collagen I in EPDCs at the annulus fibrosis. Blue arrowheads indicate pro-collagen I negative EPDCs within myocardium. Bar = 50 μm.

Figure 6. EMT gene expression profile of annulus fibrosis EPDCs

(A) Schematic figure showing the dissection of the atrioventricular junction region, dissociation of the region including annulus fibrosis EPDCs, and subsequent FACS isolation of EPDCs for qRTPCR analysis. (B) AF EPDCs were low in cardiomyocyte specific gene expression (Myh6, Tnnt2). (C–E) At E14.5, AF EPDCs were enriched for mesenchymal and fibrosis transcripts (C), EMT-associated transcripts (D), and transcripts encoding the matrix degrading enzyme Mmp2 (E). (F–G) Continued and further upregulated expression of fibrosis, mesenchymal, and EMT-associated transcripts at E15.5-E16.5. n=3. *P < 0.05. Dotted line indicate gene expression of non-EPDCs (GFP- population), assigned a value of 1.

Figure 7. Gene expression signature of Wt1 epicardial progenitors

(A) Schematic showing dissociation and FACS isolation of GFP⁺ epicardial cells from Wt1^GFPCre/+ E14.5-E15.5 hearts for qRTPCR analysis. GFP (green) was specifically expressed in epicardium. White arrows indicate colocalization with epicardium marker WT1 (red). Bar = 50 μm. (B) Gene expression signature of GFP+ epicardial cells. n=3–4. *P < 0.05. Dotted line indicates gene expression of GFP- non-epicardial cells, assigned a value of 1.

Figure 8. Gene expression signature of AF versus apical EPDCs

(A) Schematic showing the dissociation and FACS isolation of AF and apex EDPCs from Wt1^CreERT2/+ Rosa26^mTmG/+E15.5 hearts for qRTPCR analysis. (B) Many of the fibrosis and EMT-associated trascripts were significantly upregulated in AF vs apex EPDCs. n=3–4. *P < 0.05. Dotted line indicates gene expression of apical EPDCs, assigned a value of 1.