Rapid upregulation and clearance of distinct circulating microRNAs after prolonged aerobic exercise

Abstract

Short nonprotein coding RNA molecules, known as microRNAs (miRNAs), are intracellular mediators of adaptive processes, including muscle hypertrophy, contractile force generation, and inflammation. During basal conditions and tissue injury, miRNAs are released into the bloodstream as "circulating" miRNAs (c-miRNAs). To date, the impact of extended-duration, submaximal aerobic exercise on plasma concentrations of c-miRNAs remains incompletely characterized. We hypothesized that specific c-miRNAs are differentially upregulated following prolonged aerobic exercise. To test this hypothesis, we measured concentrations of c-miRNAs enriched in muscle (miR-1, miR-133a, miR-499-5p), cardiac tissue (miR-208a), and the vascular endothelium (miR-126), as well as those important in inflammation (miR-146a) in healthy male marathon runners (N = 21) at rest, immediately after a marathon (42-km foot race), and 24 h after the race. In addition, we compared c-miRNA profiles to those of conventional protein biomarkers reflective of skeletal muscle damage, cardiac stress and necrosis, and systemic inflammation. Candidate c-miRNAs increased immediately after the marathon and declined to prerace levels or lower after 24 h of race completion. However, the magnitude of change for each c-miRNA differed, even when originating from the same tissue type. In contrast, traditional biomarkers increased after exercise but remained elevated 24 h postexercise. Thus c-miRNAs respond differentially to prolonged exercise, suggesting the existence of specific mechanisms of c-miRNA release and clearance not fully explained by generalized cellular injury. Furthermore, c-miRNA expression patterns differ in a temporal fashion from corollary conventional tissue-specific biomarkers, emphasizing the potential of c-miRNAs as unique, real-time markers of exercise-induced tissue adaptation.

Keywords: cardiorespiratory fitness biomarker; cardiovascular biomarker; circulating microRNA; exercise physiology; prolonged aerobic exercise.

Fig. 1.

Baseline characteristics of candidate circulating microRNAs (c-miRNAs). A: under resting conditions before marathon run, c-miR-1, c-miR-133a, c-miR-208a, and c-miR-134 were nearly undetectable in plasma, whereas c-miR-146a and c-miR-126 were detectable at higher relative levels. Baseline steady-state c-miRNA levels displayed a relatively modest degree of variability among the athletes. Data are presented as box and whisker plots signifying copy numbers of c-miRNA expression levels (see materials and methods), where horizontal lines denote median, boxes denote 25% and 75% percentile confidence intervals, and error bars reflect maximum and minimum values. All plots are labeled with statistical medians (1st quartile, 3rd quartile). B: modest alterations in brain-enriched c-miR-134 in the context of prolonged aerobic exercise. c-miR-134 was poorly expressed at rest, but was modestly upregulated after the end of the run with a subsequent decrease at 24 h postmarathon. C: similar to the pattern of regulation for c-miR-134, modest alterations in the endothelial-specific c-miR-126 were observed after the end of the run, followed by a decrease at 24 h postmarathon. For each marathon athlete, baseline biomarker levels under resting condition are assigned a fold change of 1, to which measurements obtained during subsequent study time points (i.e., shortly after and 24 h after prolonged aerobic exercise) are compared. Data are represented as box and whisker plots signifying fold changes in c-miRNA levels and are labeled with statistical medians, interquartile range (IQR), and P values of comparisons between conditions. PRE, the day before the marathon; FINISH, immediately after completion of the marathon; POST-24, the day following the marathon within 24 h of finishing time.

Fig. 2.

Distinct muscle-specific c-miRNAs are elevated after prolonged aerobic exercise and are downregulated 24 h postexercise. c-miR-1 (A) and c-miR-133a (B) were increased after prolonged aerobic exercise and returned very close to baseline by POST-24. C: in contrast, plasma CPK levels increased after exercise and remained elevated POST-24. D: c-miR-499–5p was increased after prolonged aerobic exercise and displayed a mixture of elevated and nonelevated measurements by POST-24. In contrast with c-miR-1 or c-miR-133a, the magnitude of increase of c-miR-499–5p was modest and was more similar to the profiles of c-miR-134 (Fig. 1B) or c-miR-126 (Fig. 1C). For each marathon athlete, baseline biomarker levels under resting condition are assigned a fold change of 1, to which measurements obtained during subsequent study time points (i.e., shortly after and 24 h after prolonged aerobic exercise) are compared. In all panels, data are represented as box and whisker plots signifying fold changes in c-miRNA levels, where horizontal lines denote medians, boxes denote 25% and 75% percentile confidence intervals, and error bars reflect maximum and minimum values. All panels are labeled with statistical medians, IQR, and P values of comparisons between conditions.

Fig. 3.

Cardiac muscle-enriched c-miR-208a is elevated after prolonged aerobic exercise and is downregulated 24 h postexercise. A: c-miR-208a increased significantly after prolonged exercise and decreased nearly 50-fold by POST-24 to levels not significantly different from baseline. B: troponin I levels increased after prolonged aerobic exercise. However, despite falling significantly in the time period at FINISH and POST-24, troponin I levels remained significantly elevated POST-24 compared with prerun. C: ultrasensitive troponin I levels were significantly upregulated and increased after exercise but only subtly decreased POST-24. D: NH₂-terminal prohormone of brain natriuretic peptide (NT-proBNP) levels were also significantly increased postexercise, but exhibited only a slight, nonstatistically significant decrease in the 24 h following completion of exercise. Thus levels at 24 h postexercise were still significantly higher than preexercise. In A–D, for each marathon athlete, biomarker levels are presented in a similar manner to that of Fig. 1B. All panels are labeled with statistical medians, IQR, and P values of comparisons between conditions.

Fig. 4.

Inflammation-specific c-miR-146a is elevated after prolonged aerobic exercise and downregulated 24 h postexercise. A: c-miR-146a increased significantly following prolonged aerobic exercise and nearly returned to baseline POST-24. B: high-sensitivity C-reactive protein (hsCRP), however, was not altered at the end of run, but substantially increased POST-24. A and B: for each marathon athlete, biomarker levels are presented in a similar manner to that of Fig. 1B. All panels are labeled with statistical medians, IQR, and P values of comparisons between conditions.

Fig. 5.

Muscle-specific and cardiac-specific miRNAs are elevated in circulating plasma after prolonged aerobic exercise, but not after acute exhaustive exercise. A, top: the plasma of 11 athletes (N = 11) was analyzed in conjunction with acute exhaustive exercise. At baseline resting conditions before acute exhaustive exercise, c-miR-1 was poorly expressed. At the end of exercise, c-miR-1 did not show any significant alterations in expression. Bottom: in 21 marathon athletes (N = 21) analyzed for this study, c-miR-1 levels were significantly increased at FINISH and returned to baseline by POST-24. B, top: c-miR-208a expression was at the level of detectability at baseline resting conditions and following acute exhaustive exercise (N = 11). Bottom: in marathon runners, c-miR-208a levels were significantly upregulated at FINISH and returned to baseline by POST-24 (N = 21). Data are presented as box and whisker plots signifying fold changes in c-miRNA levels, where horizontal lines denote median, boxes denote 25% and 75% percentile confidence intervals, and error bars reflect maximum and minimum values. Baseline biomarker levels under resting condition are assigned a fold change of 1, to which measurements obtained during subsequent study time points (i.e., FINISH and POST-24) are compared. All panels are labeled with statistical medians, IQR, and P values of comparisons between conditions.