Proteomic characterization of epicardial-myocardial signaling reveals novel regulatory networks including a role for NF-κB in epicardial EMT

<div><p>Signaling between the epicardium and underlying myocardium is crucial for proper heart development. The complex molecular interactions and regulatory networks involved in this communication are not well understood. In this study, we integrated mass spectrometry with bioinformatics to systematically characterize the secretome of embryonic chicken EPDC-heart explant (EHE) co-cultures. The 150-protein secretome dataset established greatly expands the knowledge base of the molecular players involved in epicardial-myocardial signaling. We identified proteins and pathways that are implicated in epicardial-myocardial signaling for the first time, as well as new components of pathways that are known to regulate the crosstalk between epicardium and myocardium. The large size of the dataset enabled bioinformatics analysis to deduce networks for the regulation of specific biological processes and predicted signal transduction nodes within the networks. We performed functional analysis on one of the predicted nodes, NF-κB, and demonstrate that NF-κB activation is an essential step in TGFβ2/PDGFBB-induced cardiac epithelial-to-mesenchymal transition. In summary, we have generated a global perspective of epicardial-myocardial signaling for the first time, and our findings open exciting new avenues for investigating the molecular basis of heart development and regeneration.</p></div

NF-κB p65 is expressed in chicken embryonic epicardium.

<p>Cryosections from HH22 (A, A’, B, B’), HH26 (C, C’, D, D’) and HH30 (E, E’, F, F’) chick embryos were processed for immunohistochemistry using antibodies specific for p65 (red) and anti-sarcomeric myosin (MF20). The cardiomyocyte marker MF20 (green) labels the myocardium. Nuclei are detected by DAPI (blue). Rectangles mark the regions that are magnified in the lower panel for each stage. Scale bar: 50 μm for (A’, B’, C’, D’, E’, F’); 315 μm for (A, B, C, D, E, F).</p

Primers used in qRT-PCR.

<p>Primers used in qRT-PCR.</p

Proposed model for the function of NF-κB signaling in epicardial EMT.

<p>TGFβ2 and PDGFBB induce the activation of epicardial NF-κB and consequently upregulation of mesenchymal genes <i>MMP2</i> and <i>DCN</i> as well as downregulation of epicardial cell marker gene <i>WT1</i>.</p

NF-κB p65 subcellular localization in primary mouse epicardial cells after TGFβ2 and PDGFBB treatment.

<p>Immunofluorescent detection of p65 in untreated (UTD) cells (A-D), cells treated with TGFβ2/PDGFBB for 8 hours (E-H), and cells co-treated with TGFβ2/PDGFBB and BMS345541 (I-L). Arrows point to untreated cells with elevated nuclear p65 staining as compared to cytoplasmic staining levels. Scale bar: 20 μm. Box-and-Whisker plots (Min to Max method, Graphpad Prism) of p65 nuclear:cytoplasmic ratio (M). *p < 0.0001 (Mann—Whitney test).</p

Inhibition of NF-κB blocks TGFβ2- and PDGFBB-induced EMT in the mouse epicardial cell line MEC1.

<p>MEC1 cells cultured in the absence or presence of TGFβ2/PDGFBB and/or BMS345541 for 48 hours. Immunodetection of F-actin (A-D) and β-catenin localization (A’-D’) in the same cells. In addition, expression of ZO1 (E-H) and ACTA2-positive stress fiber formation (E’-H’) was determined in the same cells. Scale bar: 40 μm.</p

NF-κB p65 is expressed in mouse embryonic epicardium.

<p>Immunofluorescence imaging of mouse E11.5 (A-B) and E13.5 (C-D) sections using antibodies directed against p65 (red) and cTnT (green). Nuclei identified by DAPI (blue) staining. p65 proteins were found enriched in the epicardium and endocardium. Nuclear accumulation was observed in a subset of epicardial cells (arrows). Dotted lines delineate the border between the myocardium and the epicardium. Scale bars: 20 μm.</p

The NF-κB regulatory network.

<p>Ingenuity Pathway Analysis was utilized to generate network connections of the EHE secretome. In the depicted network NF-κB was identified as a central node. Nodes are depicted as molecule names over shapes. Shaded shapes represent focus molecules and open shapes represent molecules were not present in our dataset but placed by IPA due to their connections to the focus molecules. Single lines represent protein—protein interactions and arrows indicate regulatory relationship(s) between connected molecules. Solid lines denote a direct relationship between gene products while dotted lines indicate an indirect relationship. TGFβ (circle) and NF-κB (square) connectivity nodes are marked.</p

IPA-predicted functional networks in the EPDC-heart explant secretome.

<p>IPA-predicted functional networks in the EPDC-heart explant secretome.</p