1Universitätsklinikum Düsseldorf Institut für Herz- und Kreislaufphysiologie Düsseldorf, Deutschland; 2Heinrich-Heine-Universität Düsseldorf Biologisch-Medizinisches Forschungszentrum (BMFZ) Düsseldorf, Deutschland
Pericardium is known as a fibrous sac that protects the heart from excessive filling and secretes pericardial fluid serving as a lubricant to reduce friction between the heart and surrounding organs. Moreover, its visceral layer (epicardium) is a source of WT1+ progenitor cells and paracrine factors during embryonic development and in adulthood after activation in response to myocardial infarction (MI). Due to the fact that its adjacent parietal and fibrous layers were barely studied, we aimed to examine the pericardium, its cellular composition and response to MI.
Histological analysis of murine pericardium, showed that it is a complex structure, which, apart from its sublayers, contains fat depots composing up to 60%±8.9 of total pericardial area, and fat-associated lymphoid clusters (FALCs) accounting for 8.7%±5.8 of pericardial fat depots area.
To analyze cellular composition, we used immune fluorescence and a lineage-tracing model that allows inducible expression of the tomato protein under WT1 promoter control (WT1CreERT2;Rosa26Tomato), where WT1 (Wilms' tumor 1) is known as an epicardial progenitor cell marker. Interestingly, both approaches demonstrated that in contrast to the postnatal epicardium, pericardium contained a multitude of WT1+ cells already under basic condition. This observation was confirmed by qPCR analysis of separately isolated pericardium and cultivated pericardial cells. In addition to WT1+ cells, we detected CD68+ and F4/80+ macrophages, CD19+ B-cells and CD31+ endothelial cells located within fat depots and FALCs.
To examine a pericardial response to MI we conducted a surgical induction of ischemia/reperfusion (I/R) injury in a mouse model with closed pericardium and analyzed the tissue on day 6 after the surgery. Pericardial thickness increased from 10µm±3 (control) to 147µm±45 (scar area) and 17µm±14 (remote area) after I/R. Importantly, after sham the thickness expanded up to 113µm±14 and 12µm±0,3 along the left and right ventricles respectively. This change was accompanied by presence of Ki67+ proliferating cells located in all pericardial elements. Moreover, we observed a 3-fold increase in FALCs number and found integration of adipocytes into FALCs as well as in the adjacent parietal and fibrous layers.
Interestingly, histological analysis of pericardium from WT1CreERT2;Rosa26Tomato mice showed that after I/R injury Tomato+ cells are detectable not only on the parietal and fibrous layers but also on the epicardium and within the scar area, what may indicate a migration of activated pericardial WT1+ derived cells towards the injured heart.
Single-cell RNA-sequencing analysis of pericardium under basic condition, after sham and I/R surgery revealed 20 different cell clusters. I/R induced mainly an increase in B-cells, whereas sham promoted a myeloid cell response indicating a differential response to damage. Surprisingly, there were two separated clusters of WT1+ cells, one expressing mesothelial cell markers (Upk3b, MSLN, Lrrn4) and the other rather fibroblast markers (Tgfb2, Ddr2, Postn).
We have shown that pericardium is an intricate structure composed of various cell types. Its ability to be activated along with the upregulation of immune cells within FALCs suggests that it may modulate cardiac inflammation and repair upon injury. The presence of WT1+ cells raises the possibility that pericardium is also a source of cells, able to differentiate, migrate and contribute to the scar remodeling process.