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. 2020 Jun 10:11:605.
doi: 10.3389/fphys.2020.00605. eCollection 2020.

Sedentary and Trained Older Men Have Distinct Circulating Exosomal microRNA Profiles at Baseline and in Response to Acute Exercise

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Sedentary and Trained Older Men Have Distinct Circulating Exosomal microRNA Profiles at Baseline and in Response to Acute Exercise

Venugopalan D Nair et al. Front Physiol. .

Abstract

Exercise has multi-systemic benefits and attenuates the physiological impairments associated with aging. Emerging evidence suggests that circulating exosomes mediate some of the beneficial effects of exercise via the transfer of microRNAs between tissues. However, the impact of regular exercise and acute exercise on circulating exosomal microRNAs (exomiRs) in older populations remains unknown. In the present study, we analyzed circulating exomiR expression in endurance-trained elderly men (n = 5) and age-matched sedentary males (n = 5) at baseline (Pre), immediately after a forty minute bout of aerobic exercise on a cycle ergometer (Post), and three hours after this acute exercise (3hPost). Following the isolation and enrichment of exosomes from plasma, exosome-enriched preparations were characterized and exomiR levels were determined by sequencing. The effect of regular exercise on circulating exomiRs was assessed by comparing the baseline expression levels in the trained and sedentary groups. The effect of acute exercise was determined by comparing baseline and post-training expression levels in each group. Regular exercise resulted in significantly increased baseline expression of three exomiRs (miR-486-5p, miR-215-5p, miR-941) and decreased expression of one exomiR (miR-151b). Acute exercise altered circulating exomiR expression in both groups. However, exomiRs regulated by acute exercise in the trained group (7 miRNAs at Post and 8 at 3hPost) were distinct from those in the sedentary group (9 at Post and 4 at 3hPost). Pathway analysis prediction and reported target validation experiments revealed that the majority of exercise-regulated exomiRs are targeting genes that are related to IGF-1 signaling, a pathway involved in exercise-induced muscle and cardiac hypertrophy. The immediately post-acute exercise exomiR signature in the trained group correlates with activation of IGF-1 signaling, whereas in the sedentary group it is associated with inhibition of IGF-1 signaling. While further validation is needed, including measurements of IGF-1/IGF-1 signaling in blood or skeletal muscle, our results suggest that training status may counteract age-related anabolic resistance by modulating circulating exomiR profiles both at baseline and in response to acute exercise.

Keywords: Acute aerobic exercise; aging; insulin growth factor-1 signaling; microRNA profiling; plasma exosomes; regular endurance training.

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Figures

FIGURE 1
FIGURE 1
Characterization of exosome-enriched preparations isolated from plasma and analysis of their small RNA contents. (A) Analysis of EV size distribution and concentration in exosome-enriched preparations using a NanoSight NS300 instrument. (B) Representative image from the NanoSight instrument showing optimal light scatter from plasma-derived nanovesicles. (C,D) Bioanalyzer traces of the small RNAs extracted from exosome-enriched preparations following incubation with DNase or RNase. Lane 1, Control (non-treated); lane 2, DNase-treated; lane 3, RNase-treated. (E) Sequencing analysis of the small RNAs extracted from exosome-enriched preparations. Blood was drawn from each study participant at 3 time points: before exercise (Pre; open bars), immediately after exercise (Post; diagonal line bars), and 3 h after exercise (3 hPost; vertical line bars). Participants comprised groups of older trained and sedentary adults, as indicated. Shown at the top are the total number of reads, the percentage of mappable reads after adapter clipping, and the percentage of reads mapping to the human genome/transcriptome. Stacked-bar plots represent the percentage of mapped reads assigned to miRNA, rRNA, tRNA, other gencode transcripts (which are not miRNA, rRNA or tRNA), and other mapped sequences (mapped to the genome, but not to the four transcript types mentioned above). The different RNA species are color-coded. Shown at the bottom is the percentage of transcriptome complexity, defined as the ratio of the number of unique transcript sequences to the total number of transcript sequences.
FIGURE 2
FIGURE 2
Differential expression of circulating exomiRs at baseline in the trained and sedentary groups. (A) Heat map with dendrogram illustrating clustering results for the 32 exomiRs identified as differentially expressed when comparing miRNA expression changes between groups (sedentary vs. trained) at baseline (Pre) and between time points (Post vs. Pre and 3hPost vs. Pre) within each group. Hierarchical clustering was applied using the average linkage method and Euclidean distance. Values signify the log2-transformed cpm (counts per million reads of the library size) normalized within each row such that the mean is zero and the standard deviation is one. Colors represent the level of miRNA expression; red: high expression; green: low expression. Groups numbered from 1 to 4 on the right highlight exomiRs that were differentially expressed at baseline (1 and 2) and at Post (4) between sedentary and trained subjects, and those differentially expressed at either Post or 3 hPost relative to Pre in trained subjects (3). (B) Differential expression of circulating exomiRs in the trained relative to sedentary group at baseline (Pre). The p-value in log10 scale of each miRNA is plotted against its fold change in log2 scale and each circle denotes a miRNA. The miRNAs with p-values ≤ 0.05 and log2 (fold change) ≥ 1 in either direction is represented by either red circles (upregulated) or green circles (downregulated). Three circulating exomiRs were upregulated and one was downregulated in the trained compared to sedentary group.
FIGURE 3
FIGURE 3
Differential regulation of circulating exomiRs following acute exercise in the trained and sedentary groups. (A) Differentially regulated circulating exomiRs immediately after acute exercise and 3 h post-exercise (B) compared to baseline (Pre) in trained group. (C) Differentially regulated circulating exomiRs immediately after exercise and 3 h post-exercise (D) compared to baseline in sedentary group. The p-value in log10 scale of each miRNA is plotted against its fold change in log2 scale and each circle symbolizes a miRNA. The miRNAs with p-values ≤ 0.05 and log2 (fold change) ≥ 1 in either direction are represented by either red circles (upregulated) or green circles (downregulated). In (A) three miRNAs were upregulated and four were downregulated. In (B) five miRNAs were upregulated and three were downregulated. In (C) eight miRNAs were upregulated and one was downregulated. In (D) all four miRNAs were downregulated.
FIGURE 4
FIGURE 4
Acute exercise regulates distinct circulating exomiRs in the trained and sedentary groups. (A) Venn diagram depicting the exomiRs that were differentially regulated at Post and at 3 hPost in the trained group (7 and 8, respectively) and in the sedentary (9 and 4, respectively). There is no overlap between the trained and sedentary groups at either time point. (B) and (C) Shown are the expression levels of miR-383-5p (B) and miR-505-3p (C) in the circulating exosomes of sedentary and endurance-trained subjects before exercise (Pre), immediately after exercise (Post), and 3 h after exercise (3 hPost). Bar plots represent log2-transformed normalized read counts from small RNA sequencing.
FIGURE 5
FIGURE 5
Association of circulating exomiR signatures with IGF1 signaling. Shown are the circulating exomiRs that were differentially regulated at Post, along with their reported/predicted target genes linked to IGF1 signaling, when applicable. Red border-rectangles indicate miRNAs that are upregulated; blue border-rectangles indicate miRNAs that are downregulated. Colors represent the level of miRNA expression; red: high expression; green: low expression. Up/down arrows indicate whether a target gene is up- or downregulated, respectively. (A) Circulating exomiRs that are differentially expressed in exercise-trained individuals relative to the sedentary lead to inhibition of IGF1 signaling. (B) In trained individuals, acute exercise-regulated exomiRs increase tumor suppressor activity and activate IGF1 signaling. (C) In sedentary individuals, the majority of acute exercise-regulated exomiRs are upregulated and result in downregulation of IGF1 signaling. Note that downregulated miR-4433b-3p is not shown because its target is undetermined.

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