From moderate to strenuous training: unravelling mechanistic contributors and biomarkers for atrial fibrillation in exercise

Figures

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  Figure 1. Results of the electrophysiological study. (A) Recordings of burst pacing atrial fibrillation (AF) induction in SED, moderately trained (MOD), and intensively trained (INT) rats (in each group: ECG in the superior panel, intracavitary electrogram in the middle, and stimulus artefact in the lower panel). Cycle length for burst pacing was 40 ms in SED and INT, and 60 ms in MOD. (B) AF inducibility rate in the SED (n = 21), MOD (n = 20), and INT (n = 18) groups as a percentage of rats >1 s inducible AF (P for trend = 0.009). (C) AF duration (boxplot) of the irregular atrial electrograms in seconds. (D–F) Measurements (boxplot) of the electrophysiological parameters WCL (D; n = 20 SED, n = 19 MOD, n = 17 INT), ARP (E; n = 19 SED, n = 19 MOD, n = 16 INT), and cSNRT (F; n = 20 SED, n = 18 MOD, n = 17 INT). Most analyses were performed with one-way ANOVA, which was significant at P < 0.05. Post hoc comparisons were performed with the least significant difference (LSD) test. Induced-AF duration was analysed with a Kruskal–Wallis test and post hoc Dunn test. *P < 0.05, **P < 0.01, and ***P < 0.001. AF, atrial fibrillation; ARP, atrial effective refractory period; cSNRT, corrected sinus…

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  Figure 2. Results of the electrocardiographic analysis. (A) Representative averaged ECG recordings (thin green tracings represent 200 consecutive beats, the thick black trace is the averaged ECG recording) of a rat in the SED, MOD, and INT exercise groups. (B–F, H) Results (boxplot) and comparison of the RR interval (B), P duration (C), PR interval (D), QRS (E), and QTc duration (F). For B to F: n = 18 SED, n = 20 MOD, n = 18 INT. Analyses with a one-way ANOVA, which was significant at the P < 0.05 level for the RR interval, P duration, and LF/HF ratio. Post hoc comparisons with group levels (LSD test) are shown. (G) Representative images of the LF/HF peaks for a rat in each group and LF/HF ratio measurements (H). n = 16 SED, n = 17 MOD, n = 17 INT. *P < 0.05, **P < 0.01. ECG, electrocardiogram; HF, high frequency; INT, high-intensity; LF, low frequency; MOD, moderate; SED, sedentary groups.

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  Figure 3. Structural remodelling assessment in all groups. (A) Measurement of echocardiographic images of the left atrial diameter (LAD, longitudinal parasternal view) and the right (RA) and left atrial (LA) areas (apical 4-chamber view). RV, right ventricle; LV, left ventricle; Ao, aorta. (B, C) Atrial echocardiographic measurements (boxplot). Indexed LAD (B) was compared with one-way ANOVA, which was significant at the P < 0.001 level, and post hoc comparisons between group levels [least significant difference test (LSD)] were performed. N = 15, 20 and 15 for SED, MOD, and INT, respectively. Indexed left (n = 10, 13, 15 for SED, MOD, INT) and RA (n = 14, 16, 17 for SED, MOD, INT) areas (C) were compared with linear mixed-effects modelling, including group, atrium and their interaction as dependent variables; the group and atrial factors were significant at the P < 0.05 and P < 0.001 levels, respectively. Post hoc comparisons between group levels (LSD) are shown. (D) Representative microphotographs of atrial specimens from all groups stained with Sirius red. The scale corresponds to 100 µm. (E) Fibrosis quantification (mean ± SEM) in the LA and RA (n = 9, 8, 8 for SED, MOD, INT). Analyses…

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  Figure 4. Transcriptomic analysis. (A, C) Volcano plot of the moderate (MOD) vs. intensive (INT) trained group comparison in the left atrium (LA; MOD n = 8, INT n = 8) (A) and right atrium (RA; MOD n = 7, INT n = 8) (C). In each atrium, dark blue points (right side of Volcano plots) represent genes significantly up-regulated (fold-change > 1.5) in the INT group compared with MOD, light blue points (left side of Volcano plots) represent genes significantly up-regulated (fold-change > 1.5) in the MOD group compared with INT, and grey points are genes not differentially expressed between MOD or INT or with a fold-change <1.5. (B, D) Overrepresentation analysis of the differentially expressed genes (DEGs) in WikiPathways in the LA (B) and RA (D) of the MOD vs. INT comparison. (E) Genes deregulated in the cytokines and inflammatory response according to the WikiPathway analysis.

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  Figure 5. Assessment of local inflammation. (A) Representative images of immunochemical staining of CD68-positive cells (brown) in the left (LA) and right (RA) atria. The scale corresponds to 50 µm. (B) Number of CD68-positive cells per square millimetres in the LA and RA (mean ± SEM). N = 9, 7, 8 for SED, MOD, INT, respectively. Two-way ANOVA was performed, with group, atrium and their interaction as dependent variables. The group × atrium interaction was significant at the P < 0.01 level; within each atrium, significant post hoc pairwise comparisons between groups [least significant difference test (LSD)] are shown. (C) Correlation between the degree of atrial fibrosis and the presence of CD68-positive inflammatory cells. Analysis with a linear mixed model including CD68 infiltration, atria, and their interaction as predictors in a model with a repeated measurements covariate structure. Atria × CD68 infiltration was significant at the P < 0.05 level; the slope at every atrium was subsequently tested and is shown. The CD68 infiltration dependent variable was log-transformed to fulfil the normality of the residual requirement. *P < 0.05, **P < 0.01, ****P < 0.0001. INT, high-intensity;…

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  Figure 6. Plasmatic biomarker profile in trained groups after exercise. (A) Plasma proteins showing a significant group × timepoint interaction [e.g. different behaviours after MOD and intensive (INT) exercise] are coloured vs. the –log(P-value); dot size is inverse to P-value. (B) Cytokine, chemokine, and growth factor boxplots of altered proteins analysed in plasma. The results were analysed with a linear mixed model with a repeated measures covariance structure including group (MOD and INT) and timepoint (at rest, post-exercise), and their interaction as independent variables; all interactions were significant (see panel A). Post hoc comparisons are shown. N = 7 for MOD, n = 4 for INT at both timepoints. (C) Plasmatic biomarkers significantly increased after exercise and in the pathological pressure overload model (boxplots). For the pathological model (left panels), the results were analysed with a t-test. *P < 0.05, **P < 0.01, ****P < 0.0001 basal vs. post-exercise within each group; #P < 0.05, ##P < 0.01, ##P < 0.001 MOD vs. INT within time-point. INT, high-intensity; MOD, moderate; SED, sedentary groups.