Single-nucleus transcriptomics of epicardial adipose tissue from females reveals exercise control of innate and adaptive immune cells

Abstract

Coronary artery disease (CAD) is a leading cause of death in women. Although exercise mitigates CAD, the mechanisms by which exercise impacts epicardial adipose tissue (EAT) are unknown. We hypothesized that exercise promotes an anti-inflammatory microenvironment in EAT from female pigs. Yucatan pigs (n=7) were assigned to sedentary (Sed) or exercise (Ex) treatments and coronary arteries were occluded (O) with an ameroid to mimic CAD or remained non-occluded (N). EAT was collected for bulk and single nucleus transcriptomic sequencing (snRNA-seq). Exercise upregulated G-protein coupled receptor, S100 family, and FAK pathways and downregulated the coagulation pathway. Exercise increased the interaction between immune, endothelial, and mesenchymal cells in the insulin-like growth factor pathway and between endothelial and other cell types in the platelet endothelial cell adhesion molecule 1 pathway. Sub-clustering revealed nine cell types in EAT with fibroblast and macrophage populations predominant in O-Ex EAT and T cell population predominant in N-Ex EAT. Coronary occlusion impacted the largest number of genes in T and endothelial cells. Genes related to fatty acid metabolism were the most highly upregulated in non-immune cells from O-Ex EAT. Sub-clustering of endothelial cells revealed that N-Ex EAT separated from other treatments. In conclusion, aerobic exercise increased interaction amongst immune and mesenchymal and endothelial cells in female EAT. Exercise was minimally effective at reversing alterations in gene expression in endothelial and mesenchymal cells in EAT surrounding occluded arteries. These findings lay the foundation for future work focused on the impact of exercise on cell types in EAT.

Keywords: Epicardial adipose tissue; Major classification is Biological Sciences; Minor classification is Physiology; aerobic exercise; coronary artery disease; immune cells; swine.

Figure 1.. Schematic representation of the experimental protocol followed during the experiment.

(A) Overall animal study design (B) exercise training regimen (C) Heart to body weight ratio and citrate synthase in female pigs at the end of the experiment (D) Bulk and single nuclei RNA extraction and computational analysis. This image was created using BioRender. CS: citrate synthase; HW: heart weight; BW: body weight; LCX: left circumflex coronary artery; EAT: epicardial adipose tissue; DE: differentially expressed. n=4 per treatment group; * p < 0.05.

Figure 2.. Pathways and diseases in epicardial adipose tissue of female pigs affected by exercise.

(A) Top 30 pathways found in bulk adipose tissue to be significantly affected by exercise in female pigs. Orange: predicted upregulation; Blue: predicted downregulation; White: no effect. (B) Heat map of diseases and processes in bulk adipose tissue to be significantly affected by exercise training in female pigs. Orange: increased effect; Blue: decreased effect; White: no effect. (C) Estrogen receptor alpha network in bulk adipose tissue impacted by exercise. (D) Insulin signaling network in bulk adipose tissue impacted by exercise. (E) Predicted regulators of smooth muscle cell proliferation in bulk adipose tissue affected by exercise. Green: decreased gene expression; Red: increased gene expression; Blue: predicted gene inhibition; Orange: predicted gene activation

Figure 3.. Single nuclei gene expression from epicardial adipose tissue of sedentary and exercised female pigs.

(A) Single nuclei clustering in epicardial adipose tissue from exercise-trained and sedentary female pigs. (B) Expression of established marker genes for each cell type in each cluster. Circle plots representing most prominent cell-cell communications between exercise and sedentary groups in the insulin-like growth factor (IGF) (C), neural cell adhesion molecule (NCAM) (D), and platelet endothelial cell adhesion molecule 1 (PECAM1) (E) signaling pathways.

Figure 4.. Single nuclei gene expression from epicardial adipose tissue surrounding occluded and non-occluded coronary arteries from sedentary and exercise trained female pigs.

(A) Occluded and non-occluded unbiased clustering of 24,382 cells revealed nine different cell clusters. (B) Number and percentage of cell types in each treatment (exercise trained and sedentary) and batch-wise distribution in each occlusion*exercise treatment (occluded and non-occluded) expressed genes. (C) Sub-clustering of endothelial cells showed separation of N-Ex group from all other treatments. (D) Sub-clustering of mesenchymal cells showed four different cell types. (E) Percentage of each cell type in the mesenchymal subcluster by occlusion*exercise treatment. N: non-occluded; O: occluded; Ex: exercise trained; Sed: sedentary.