Impact of NSAIDs and endurance exercise on myocardial fibrosis and arrhythmogenesis in murine coxsackieviral myocarditis

Abstract

Athletes often exhibit unexplained non-ischemic myocardial fibrosis, which is associated with malignant arrhythmias. Given the prevalent (over)use of NSAIDs among athletes and their harmful effects during viral myocarditis, this study examined the effects of combined NSAIDs and endurance exercise on the disease progression. To this end, male C57BL/6J mice underwent eight weeks of treadmill running (60 min/day; 18 cm/s) or no exercise. After two weeks, mice were implanted with mini-pumps delivering ibuprofen (70 mg/kg bw/day) or vehicle. Myocarditis was induced via intraperitoneal coxsackievirus inoculation. Mice were sacrificed six weeks post-inoculation for ventricular arrhythmogenicity evaluation and cardiac histopathological and molecular analysis. Exercising coxsackievirus-infected mice receiving ibuprofen recovered faster from weight loss. Mortality was low and similar across groups. Histopathology revealed abating inflammation and cell loss, without significant group differences. While exercise tended to increase extensive myocardial fibrosis, statistical analyses indicated no significant differences-with or without NSAIDs-in perivascular fibrosis, interstitial fibrosis, or myocardial scarring. NSAIDs-irrespective of exercise-did not increase arrhythmogenicity. In conclusion, ibuprofen in exercising mice with viral myocarditis resulted in faster weight loss recovery, without significant differences in inflammation, fibrosis, or arrhythmogenesis compared to exercise-only mice.

Keywords: Arrhythmias; Ibuprofen; Physical exercise; Ventricular fibrosis; Viral myocarditis.

Fig. 1

Body weight changes and survival. (A) Percentage change in body weight over the observation period. Data are presented as mean ± SEM. Piecewise linear regression models, with a ‘knot’ at day 6 to account for a change in slope, demonstrated significant differences in body weight changes between groups both before (P = .001) and after (P = .0001) the knot. (B) Kaplan-Meier survival analysis. Log-rank testing (Mantel-Cox) indicated no significant differences between groups (P > .999). Group sizes for panels (A,B) CVB-SED: n = 20, CVB-EEX: n = 20, CVB-NSAID-SED: n = 21, CVB-NSAID-EEX: n = 18.

Fig. 2

Myocardial inflammation at sacrifice. (A) Representative morphological images of the inflammatory lesions in the heart during the acute (D7) and chronic (D41-45) stages of coxsackievirus B3-induced myocarditis. Hematoxylin and eosin staining. Scale bar = 100 μm. (B) Semiquantitative scoring of myocardial inflammation. Monte Carlo chi-square testing indicated no significant differences between groups (P = .567). (C) Quantitative assessment of the area of inflammation in the myocardium. Kruskal-Wallis H testing revealed no significant differences between groups in the percentage of inflammation area (P = .493). (D) Cardiac CVB viral load at sacrifice was measured by dPCR. Kruskal Wallis H testing indicated no statistically significant differences between groups (P = .860). For reference, median viral load values from a subset of animals sacrificed 7 days post-inoculation are also included (P = .494). (E–G) Cardiac gene expression at sacrifice was assessed by qPCR. Messenger RNA levels are expressed as fold change relative to the CVB-SED group and normalized to GAPDH and CDK1NB. For TNFα, a two-way ANOVA with Tukey’s post-hoc test was performed, while statistical differences for IL-1β and IL-6 were evaluated using Kruskal-Wallis H testing followed by Dunn’s multiple comparisons. Data are presented as (B) frequency histograms or as (C–G) boxplots with interquartile ranges. Group sizes for panels (B–G) CVB-SED: n = 12–17, CVB-EEX: n = 10–16, CVB-NSAID-SED: n = 11–17, CVB-NSAID-EEX: n = 9–14.

Fig. 3

Myocardial fibrosis at sacrifice. (A) Representative morphological images of fibrotic lesions and scars in the heart during the chronic stage of coxsackievirus B3-induced myocarditis. Picrosirius red staining. Scale bar = 100 μm. (B) Semiquantitative scoring of myocardial fibrosis and scarring. Monte Carlo chi-square testing indicated no statistically significant differences in the extent of perivascular fibrosis (P = .273), interstitial fibrosis (P = .677), or myocardial scarring (P = .534) among the groups. (C) The myocardial scar counts and (D) collagen surface area, assessed by picrosirius red staining, were comparable between groups (P = .473 and P = .350, respectively). (E–G) Cardiac gene expression at sacrifice was assessed by qPCR. Messenger RNA levels are expressed as fold change relative to the CVB-SED group and normalized to GAPDH and CDK1NB. For genes CTGF and COL1A1, a two-way ANOVA with Tukey’s post-hoc test was performed, while statistical differences for COL3A1 were evaluated using Kruskal-Wallis H testing followed by Dunn’s multiple comparisons. Data are presented as (B) frequency histograms or as (C–G) boxplots with interquartile ranges. Group sizes for panels (B–G) CVB-SED: n = 12–17, CVB-EEX: n = 16, CVB-NSAID-SED: n = 17, CVB-NSAID-EEX: n = 14.

Fig. 4

Ventricular arrhythmias immediately prior to sacrifice. (A) Representative illustration of a stimulus during programmed electrical stimulation, showcasing a NSVT episode. (B) Arrhythmia inducibility. Log-rank (Mantel-Cox) testing revealed no significant differences between groups (P = .096). (C) Cumulative arrhythmia burden, presented as both the number of beats and the duration in milliseconds. Kruskal-Wallis H testing with Dunn’s multiple comparisons indicated no statistically significant differences between groups for both the numbers of beats (P = .066) and duration (P = .071). Data are presented as boxplots with interquartile ranges. Group sizes for panels (B,C) CVB-SED: n = 9–10, CVB-NSAID-SED: n = 8, CVB-NSAID-EEX: n = 7.