Exercise-Dependent Modulation of Immunological Response Pathways in Endurance Athletes With and Without Atrial Fibrillation

Abstract

Background: Atrial fibrillation (AF) is a common arrhythmia characterized by uncoordinated atrial electrical activity. Lone AF occurs in the absence of traditional risk factors and is frequently observed in male endurance athletes, who face a 2- to 5-fold higher risk of AF compared with healthy, moderately active males. Our understanding of how endurance exercise contributes to the pathophysiology of lone AF remains limited. This study aimed to characterize the circulating protein fluctuations during high-intensity exercise as well as explore potential biomarkers of exercise-associated AF.

Methods and results: A prospective cohort of 12 male endurance cyclists between the ages of 40 and 65 years, 6 of whom had a history of exercise-associated AF, were recruited to participate using a convenience sampling method. The circulating proteome was subsequently analyzed using multiplex immunoassays and aptamer-based proteomics before, during, and after an acute high-intensity endurance exercise bout to assess temporality and identify potential markers of AF. The endurance exercise bout resulted in significant alterations to proteins involved in immune modulation (eg, growth/differentiation factor 15), skeletal muscle metabolism (eg, α-actinin-2), cell death (eg, histones), and inflammation (eg, interleukin-6). Subjects with AF differed from those without, displaying modulation of proteins previously known to have associations with incident AF (eg, C-reactive protein, insulin-like growth factor-1, and angiopoietin-2), and also with proteins having no previous association (eg, tapasin-related protein and α₂-Heremans-Schmid glycoprotein).

Conclusions: These findings provide insights into the proteomic response to acute intense exercise, provide mechanistic insights into the pathophysiology behind AF in athletes, and identify targets for future study and validation.

Keywords: endurance athletes; exercise; lone atrial fibrillation; proteomics.

Figure 1. Schematic of study design.

Samples were collected before, during, and after high‐intensity exercise and during recovery in male endurance athletes with or without atrial fibrillation. Circulating protein changes were assessed by multiplex immunoassays or aptamer‐based proteomics, and differentially regulated proteins and pathways were identified. AF indicates atrial fibrillation; and RBC, red blood cell.

Figure 2. Plasma concentration of select endothelial and inflammatory markers across exercise time points.

Plasma concentrations of the inflammatory markers angiopoietin‐2, interleukin‐6, interleukin‐8, and soluble triggering receptor expressed on myeloid cells‐1 (A) and the endothelial markers endothelin‐1, soluble CD54/intercellular adhesion molecule‐1, sE‐Selectin, and sVCAM‐1 (B) stratified by collection time point with visual representations of mean±SD. P values for multiple group comparisons were determined by mixed‐effects analysis with Šídák's multiple comparisons test. ANGPT2 indicates angiopoietin‐2; IL, interleukin; sE‐Selectin, soluble CD62 antigen‐like family member; sICAM‐1, soluble CD54/intercellular adhesion molecule‐1; sTREM‐1, soluble triggering receptor expressed on myeloid cells 1; sVCAM‐1, soluble vascular cell adhesion molecule‐1. *, **, and *** indicate P<0.05, P<0.01, and P<0.001, respectively.

Figure 3. Pathway analysis of whole plasma aptamer‐based proteomics reveals that exercise modulates immune and angiogenic pathway sets.

A, Principal component analysis demonstrated participant‐specific clustering of samples; grouping of individual patients is represented by the dotted circles. B, Seventeen proteins were significantly differentially enriched across the 4 time points (ie, midexercise, postexercise, or recovery vs preexercise) using filtering thresholds of q<0.05 and fold change >1.5 (dark red), while 962 proteins were differentially enriched across the 4 time points using only a statistical filtering threshold of q<0.05 (light red, increased expression; light blue, decreased expression); fold changes are determined as the maximum fold change between medians of all groups and the preexercise time point. C, Selected KEGG, Reactome, INOH, PID, and BioCarta pathways significantly enriched among proteins significantly increased (q value). D, Selected KEGG, Reactome, INOH, PID, and BioCarta pathways that were significantly enriched among proteins significantly decreased across exercise (q value; between at least 2 time points). Enrichment heatmap of specific proteins throughout the course of the exercise bout scaled by Z score. E, Z scored heatmap of raw count data displaying cardiac and immunoregulatory proteins differentially expressed as a result of the exercise bout. Analyses used the preexercise time point as the referent. EPHA indicates EphA/ephrin‐A; FDR, false discovery rate; GDF‐15, growth/differentiation factor 15; IL, interleukin; INOH, Integrating Network Objects With Hierarchies; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAPK, mitogen‐activated protein kinase; MBP‐C, myosin binding protein C, cardiac; PID, Pathway Interaction Database; and TNF‐α, tumor necrosis factor‐α.

Figure 4. Pathway analysis of whole plasma aptamer‐based proteomics reveals modulated inflammatory signaling in endurance athletes with atrial fibrillation.

A, Principal component analysis demonstrated participant‐specific clustering of samples. B, All values from the 4 time points (ie, preexercise, midexercise, postexercise, recovery) in each individual were averaged. A total of 20 proteins were differentially expressed using filtering thresholds of q<0.05 and fold change >1.5 between those with AF and those without (dark red, higher in lone AF; dark blue, lower in lone AF), while 446 proteins were differentially enriched between the 2 groups, q<0.05 with no fold‐change cut‐off (light red, higher in lone AF; light blue, lower in lone AF). C, Selected KEGG, Reactome, INOH, PID, and BioCarta pathways that were significantly enriched among those proteins significantly increased across the endurance bout. D, Selected KEGG, Reactome, INOH, PID, and BioCarta pathways that were significantly enriched among those proteins significantly decreased across the endurance bout. AF indicates atrial fibrillation; FDR, false discovery rate; GPCR, G‐protein coupled receptor; JAK STAT, Janus kinase‐signal transducer and activator of transcription; IL, interleukin; INOH, Integrating Network Objects With Hierarchies; KEGG, Kyoto Encyclopedia of Genes and Genomes; and PID, Pathway Interaction Database.