Endurance exercise remodels pulmonary vein sleeve myocytes and promotes a proarrhythmic atrial substrate

Background
Atrial fibrillation (AF) susceptibility is heightened in endurance athletes but the underlying mechanisms are incompletely understood. Because pulmonary vein myocyte (PV) triggers are critical determinants of AF, we investigated PV cellular composition and transcriptional landscape in spatial context in animal models of the athlete’s heart.

Purpose
To characterize PV myocytes at the spatial resolution in trained mice and identify transcriptional changes in comparison to the sedentary animals at the single cell level.

METHODS and Results
The following experiments were performed in mice after 6 weeks of daily treadmill training (ExT) and compared to sedentary (Sed) controls. ExT mice were subjected to a 6-week intensity-controlled interval treadmill training regimen based on a protocol developed by Kemi1 et. al. We applied subcellular-resolution spatial transcriptomics to 5 μm transverse sections of formalin-fixed, paraffin-embedded (FFPE) biopsies of the mouse PV-LA junction (Figure 1A n=3 Sed and n=3 ExT preparations). We utilized standard mouse profiling panel with a 100-gene custom panel with padlock probes for a selection of transcripts relating to ion channels, inflammation and the extracellular matrix associated with AF, resulting in 450 mRNA transcript assays. This yielded 96879 individual cells across 3 Sed and 3 ExT PV preparations that passed quality control. We then used canonical cell type markers derived from published mouse PV and LA single cell RNAseq datasets2 and label transfer-based methods to collate cells into archetypical clusters (Figure 1B). Uniform Manifold Approximation and Projection (UMAP) differentiated Sed and ExT cells into 11 clusters per specimen, among them variability was registered in 3 detected cardiomyocyte cell types, myofibroblasts and fibroblasts (Figure 1C-F). Analysis of relative ion channel expression in the 3 types of cardiomyocytes (Figure 2A-D) determined that genes encoding pacemaking ion channels Hcn1, Hcn4 and Cacna1d were highly expressed in ExT PV (FDR < 0.05) (Figure 2E,G). Surprisingly, analysis of all differentially expressed transcripts revealed a striking upregulation (FDR<0.05) in many of the measured immune-inflammatory mediators (TNFa, IL-6, Cxcl1 and Nlrp3) within ExT cardiomyocytes (Figure 2F, H).

Conclusions
Our approach demonstrated that cardiomyocyte populations expressing a complement of pacemaking ion channels (Hcn1, Hcn4, Cav1.3) are increased in ExT PV. Our data indicate that ion channel changes in ExT PV myocytes, in addition to fibroblast mediated ECM deposition are involved in the conduction slowing and increased propensity to re-entry in the ExT PV-LA junction. Additionally, our discovery of enhanced pro-inflammatory signalling within ExT PV myocytes (TNFα, NLRP3, IL-6, Cxcl1) adds to a growing body of evidence supporting exploration of inflammation as a therapeutic target for AF3.