Investigation of molecular and cellular patterns of myeloid cells in human hearts with atrial fibrillation using single nucleus RNA sequencing (snRNA-Seq)

Introduction
Atrial fibrillation is the most common tachycardia disorder. If left untreated, it can lead to heart damage and stroke. Our research group investigated the development of atrial fibrillation at molecular and genetic level to gain a better understanding of the disease. The focus was on myeloid cells and their cell-cell interaction. The analysis was based on the following hypotheses: There are molecular, genetic, and cellular differences between atrial fibrillation (pAF, cAF) and the control group (SR), and between the left and right atrial appendages (LAA and RAA).

Methods
42 patients in RAA and 23 in LAA were examined. Atrial appendage tissues from three conditions were used: Sinus rhythm (SR), paroxysmal and short-term persistent atrial fibrillation (pAF), and long-term persistent and permanent atrial fibrillation (cAF). Using the single nucleus RNA sequencing method, 14 different cell types were detected in the tissue.
The difference in cellular abundance of OSM-expressing MP/Mo and aFB4.0 between the three conditions was confirmed using RNAscope.
Gene Ontology (GO) enrichment analysis is used to analyse biological processes from differentially expressed genes (DEGs).

Results
The three subtypes of myeloid cells —macrophages (MP), monocytes (Mo), dendritic cells (cDC)— were devided into 16 cell states based on their gene expression.




Fig. 1: Colouring of the UMAP according to the Leiden algorithm with annotation of cell states

For further analysis, RAA and LAA were analysed separately. Since the left atrium is the origin of atrial fibrillation, the focus was on LAA. Using centered log-ratio (CLR) transformation, it was shown that there are significant differences in cellular abundance between SR, pAF and cAF at the cell state level.
These differences vary between RAA and LAA. At cell type level, no significant differences in cellular abundance were found between conditions in myeloid cells, which makes cell state level analysis important. It was hypothesised that cell-cell interactions occur in LAA between OSM-expressing MP/Mo cell states and the fibroblast cell state (aFB4.0) in which the OSM signalling pathway is activated. This conclusion was made because the four OSM-expressing myeloid states (marked in Fig. 2) show a decrease in cellular abundance from SR to cAF, which is significant in two cases (Fig. 2). The activated OSM signalling pathway in aFB4.0 shows a similar pattern with a significant decrease from SR to cAF.





Fig. 2: CLR-transformed myeloid cell states show condition differences with SR reference

For GO analysis, the DEGs between the three conditions were determined using the Wilcoxon test.
In LAA, the comparison of pAF to SR showed an upregulation of antigen processes and presentation, an immune response to cytokines as well as leukocyte activation and differentiation. Compared to SR and pAF, in cAF more genes are expressed that play a role in actin cytoskeleton organization, cell adhesion, GTPase activity and cell motility. In RAA this classification is not yet possible.

Conclusion 
A difference between the three states as well as between RAA and LAA within myeloid cells can be seen. Detecting the interaction between OSM-expressing MP/Mo and aFB4.0 provides an indication of cell-cell interactions that cause or result from atrial fibrillation. GO analysis in LAA shows that proinflammatory processes predominate in pAF, whereas cytoskeletal processes and related cellular processes appear to be important in cAF.