Quantitative proteomics identify DAB2 as a cardiac developmental regulator that inhibits WNT/β-catenin signaling

Abstract

To reveal the molecular mechanisms involved in cardiac lineage determination and differentiation, we quantified the proteome of human embryonic stem cells (hESCs), cardiac progenitor cells (CPCs), and cardiomyocytes during a time course of directed differentiation by label-free quantitative proteomics. This approach correctly identified known stage-specific markers of cardiomyocyte differentiation, including SRY-box2 (SOX2), GATA binding protein 4 (GATA4), and myosin heavy chain 6 (MYH6). This led us to determine whether our proteomic screen could reveal previously unidentified mediators of heart development. We identified Disabled 2 (DAB2) as one of the most dynamically expressed proteins in hESCs, CPCs, and cardiomyocytes. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mutagenesis in zebrafish to assess whether DAB2 plays a functional role during cardiomyocyte differentiation. We found that deletion of Dab2 in zebrafish embryos led to a significant reduction in cardiomyocyte number and increased endogenous WNT/β-catenin signaling. Furthermore, the Dab2-deficient defects in cardiomyocyte number could be suppressed by overexpression of dickkopf 1 (DKK1), an inhibitor of WNT/β-catenin signaling. Thus, inhibition of WNT/β-catenin signaling by DAB2 is essential for establishing the correct number of cardiomyocytes in the developing heart. Our work demonstrates that quantifying the proteome of human stem cells can identify previously unknown developmental regulators.

Current Trends and Future Perspectives of State-of-the-Art Proteomics Technologies Applied to Cardiovascular Disease Research

“…As a result, candidate novel regulators of CM differentiation, for example DAB2, a negative regulator of WNT signaling, are more likely to be true hits. 36 are injected into the mass spectrometer separately ( Figure 3A). Relative changes in the abundance of each protein are estimated by either the area under the curve (AUC) of their peptides' extracted ion chromatograms, or by the number of times the proteins' peptides have been sequenced (spectral counting).…”

“…36 The researchers performed control experiments to ensure that differentiation into CMs was successful, including fluorescence-activated cell sorting for cardiomyocyte troponin to verify that the majority of cells 40 In order to catch the very early phosphorylation signaling events, the samples were analyzed between 0 and 20 min using 2 sets of triple SILAC. This kinetics analysis revealed that phosphorylation occurred as early as 1-5 min ( Figure 4D).…”

“…Consequentially, all labeled peptides appear as a single peak in the MS1 ( Figure 5A) and are thereby selected simultaneously for fragmentation. In the MS/MS scan (MS2), the tags fragment to produce 1 of 6 reporter ions with an incremental mass of 1 Da, through incorporation of an increasing number of 13 C and 15 N 36 A summary of a study that used the label-free approach to monitor the proteome during the differentiation of human embryonic stem cells (hESCs) to cardiomycytes (CMs). DAB2 was identified as a protein that associated with the intermediate cardioprogenitor cells (CPCs).…”

Endocytosis and Physiology: Insights from Disabled-2 Deficient Mice

“…Deletion of Dab2 in zebrafish embryos through Cas9 mutagenesis resulted in a significant reduction in cardiomyocyte number (Hofsteen et al, 2016), though impact of Dab2 deletion on cardiac development is not notable (Morris et al, 2002b; Moore et al, 2013). …”

Impact of miR‐26b on cardiomyocyte differentiation in P19 cells through regulating canonical/non‐canonical Wnt signalling

“…Sequential activation and inhibition through both the canonical and the non‐canonical Wnt signalling pathway is critical during the heart development. Inhibition of canonical Wnt signalling could lead to the reduction of the second heart field progenitor cells and affect the differentiation of ES cell into the cardiomyocyte . The non‐canonical Wnt signalling ligands, Wnt5a and Wnt11 express at the anterior pole to the heart tube and play roles during the second heart field migration.…”