Single-cell RNA sequencing elucidates mechanisms behind heart disease

Hubrecht researchers of the van Rooij group have demonstrated for the first time that single-cell sequencing can be used to study gene expression profiles in adult heart cells. Thanks to this method, the researchers can gain insight in the molecular mechanisms behind heart disease. The findings are published in Circulation.

Monika Gladka and colleagues studied gene expression profiles in healthy and diseased murine cardiac tissue. Consequently, that made them discover the Ckap4 protein as an important player in the activation of cells in damaged cardiac tissue.

Sequencing
To assess gene expression in the investigated heart tissue, the researchers used single-cell sequencing: a technology offering us an overview of the gene expression in individual cells. Our DNA is the blueprint for all proteins that are produced by a cell and that determine cell function. Every cell in the human body contains roughly the same DNA, but different cell types only use different parts of that common blueprint. mRNA molecules are the step between the DNA and a protein – they act as messengers, transmitting which proteins can be produced. That function makes mRNA very suitable as an indicator for cell function.

Individual cells
Until recently, the sequencing technology only allowed analyses of thousands of cells simultaneously, drawing an image of the average gene expression in these cells. Single-cell sequencing allows us to determine the gene expression profile of individual cells. First, a single cell is sorted, its mRNA is multiplied and the material receives a molecular barcode. Next, the mRNA of various cells can be combined and sequenced in the traditional way. The barcode allows researchers to trace back the data to the original cell. Therefore, these experiments give us information on the genes expressed by each individual cell.

Cardiac damage
Gladka and her colleagues applied this technique on cardiac tissue of adult mice. By comparing gene expression profiles of healthy and ischemic tissue, they discovered subpopulations of cells specific for the diseased heart. The researchers also identified the Ckap4 protein as a marker for ischemic injury – they found an elevated Ckap4 expression in activated fibroblasts from damaged cardiac tissue. That finding corresponds with the expression of other known markers in the same cells, in both murine and human diseased hearts. Gladka and colleagues confirmed the role of Ckap4 as modulator for fibroblast activation by culturing these cells in vitro and inhibiting Ckap4.

Comparison
Gladka’s research shows that single-cell sequencing is also applicable to cardiac tissue. By comparing healthy to damaged tissue, we gain insight in the molecular mechanisms of heart disease. This way, proteins are discovered that play a role in this process, like Ckap4. Mapping these proteins and their relation to heart disease might eventually lead to the discovery of new therapeutic approaches.

Gladka MM, Molenaar B, de Ruiter H, van der Elst S, Tsui H, Versteeg D, Lacraz GPA, Huibers MMH, van Oudenaarden A, van Rooij E. (2018) Single-Cell Sequencing of the Healthy and Diseased Heart Reveals Ckap4 as a New Modulator of Fibroblasts Activation. Circulation 2018; 137(8). [article]

Source – Hubrecht University

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