Small RNA-seq during acute maximal exercise reveal RNAs involved in vascular inflammation and cardiometabolic health: brief report

Abstract

Exercise improves cardiometabolic and vascular function, although the mechanisms remain unclear. Our objective was to demonstrate the diversity of circulating extracellular RNA (ex-RNA) release during acute exercise in humans and its relevance to exercise-mediated benefits on vascular inflammation. We performed plasma small RNA sequencing in 26 individuals undergoing symptom-limited maximal treadmill exercise, with replication of our top candidate miRNA in a separate cohort of 59 individuals undergoing bicycle ergometry. We found changes in miRNAs and other ex-RNAs with exercise (e.g., Y RNAs and tRNAs) implicated in cardiovascular disease. In two independent cohorts of acute maximal exercise, we identified miR-181b-5p as a key ex-RNA increased in plasma after exercise, with validation in a separate cohort. In a mouse model of acute exercise, we found significant increases in miR-181b-5p expression in skeletal muscle after acute exercise in young (but not older) mice. Previous work revealed a strong role for miR-181b-5p in vascular inflammation in obesity, insulin resistance, sepsis, and cardiovascular disease. We conclude that circulating ex-RNAs were altered in plasma after acute exercise target pathways involved in inflammation, including miR-181b-5p. Further investigation into the role of known (e.g., miRNA) and novel (e.g., Y RNAs) RNAs is warranted to uncover new mechanisms of vascular inflammation on exercise-mediated benefits on health.NEW & NOTEWORTHY How exercise provides benefits to cardiometabolic health remains unclear. We performed RNA sequencing in plasma during exercise to identify the landscape of small noncoding circulating transcriptional changes. Our results suggest a link between inflammation and exercise, providing rich data on circulating noncoding RNAs for future studies by the scientific community.

Keywords: exercise; extracellular RNA; miR-181b.

Fig. 1.

A: stacked-bar plot representing the percentage of reads assigned to various RNA species. B: volcano plot of the differentially expressed miRNAs. Green dots represent miRNAs that are differentially expressed at an absolute value (fold change) of ≥2 and false discovery rate adjusted <0.05 (Benjamini-Hochberg). C: plasma expression of miR-181b-5p in discovery and validation cohorts (P < 0.05 for validation using a two-sided Wilcoxon rank-sum test). D: top 20 canonical biological pathways from Ingenuity Pathway Analysis specified by miRNAs dysregulated during acute maximal exercise. The numbers in parentheses refer to the number of genes targeted by differentially expressed miRNAs over the total number of genes in a given pathway. The P values obtained from a two-tailed Fisher exact test were adjusted for multiple testing (Benjamini-Hochberg). E: miR-181b-5p fold change in murine skeletal muscle tissue after acute exercise in old (red; n = 3) and young (blue; n = 3) mice with respect to age-matched control (n = 3) mice as a function of time (0: before exercise; 2 and 6 h: after exercise). A two-tailed t-test was used to determine significance between the experimental group (exercised mouse) and age-matched control mice. *Bonferroni-adjusted P value < 0.05. F: miR-181b-5p expression in constituent cells in blood separated by flow sorting. Targeted quantitative PCR (qPCR) using the Qiagen miScript platform was performed, and the computed tomography (CT) values are presented as a box plot for 6 replicates of each cell type.

Fig. 2.

Differentially expressed Y RNA (A) and tRNA (B) fragments in human plasma during acute submaximal exercise, with the false discovery rate adjusted to <0.05 (Benjamini-Hochberg). The box plots represent log-transformed normalized read counts from small RNA sequencing.