Collagen mRNA Perturbations in Embryonic Chick Heart By Single Cell Sequencing

Introduction: As the most abundant protein within the animal kingdom, collagen’s role in the development of embryonic hearts is of the utmost importance. Previous studies have shown as embryonic heart develop, collagen levels increase with stiffness. This stiffness combats the mechanical stress due to the heart beating. If heart tissue damage occurs, the cells are unable to regenerate, collagen forms scar tissue and could lead to heart failure. It is likely that collagen perturbations after DNA damage occurs. In treating embryonic chicken hearts with blebbistatin (blebb), a myosin II inhibitor, the heart stops beating and there is an overall decrease in mechanical stress. This decrease in mechanical stress corresponds with lower collagen levels. This study aims to use single cell sequencing (scRNA-seq) and differential analysis of collagen genes across cell types and drug treatment groups to gain insight about collagen response to various stimuli.

Materials and Methods: Embryonic chick (Gallus gallus) hearts were extracted after 5 days of development. The chick hearts were assigned into 4 drug treatment groups: Dimethyl sulfoxide (DMSO), blebb, blebb with blebb washout(blebb+wo), and H₂O₂. DMSO and H₂O₂ serving as negative and positive control for DNA damage respectively. scRNA-seq data was analyzed using the program Seurat. All data was normalized and underwent Uniform Manifold Approximation and Projection (UMAP) to identify cell clusters. These clusters were identified using biomarkers found in literature and previous studies. Differential analysis was conducted with the default Wilcox test. Second Harmonic Generated (SHG) images visualized collagen and myosin fibers within a tissue.

Results and Discussion: Using Wilcox test differential analysis, significant changes in collagen expression were determined. All treatment groups showed significant perturbations of collagen. Compared against DMSO, Blebb+wo had a significant decrease (p<2.5e-6) in expression of 2 collagen proteins (Figure 1B-i). However, H₂O₂ treated groups had both upregulation and down regulation (p<.0002) of 2 collagen proteins (Figure 1B-ii). After clustering the scRNA-seq data with UMAP, we identified epithelial, epicardial, cardiomyocytes, macrophages, erythrocytes, and valve cells (Figure 1A). Most collagen expression was found exclusively in the valve and epicardial cells. Cardiomyocytes expressed high levels of myosin; this is further confirmed with SHG. SHG indicated a stronger forward signal, corresponding with larger amounts of myosin compared to collagen.

Conclusions: This study was successful in the identification of cell types and observing the perturbations of collagen. Further analysis should find biomarkers for DNA damage that can be seen using single cell sequencing.