MiRNA expression in human skeletal muscle following an acute bout of moderate intensity cycle ergometry exercise

Abstract

Exercise is a potent stimulus that drives both physical and metabolic adaptations, with novel emerging techniques permitting investigation of their molecular mechanisms. The purpose of this study was to utilize small RNA sequencing to determine differentially expressed miRNAs in skeletal muscle of lean humans before and following a single bout of moderate cycling exercise. Total RNA was extracted (RNeasy fibrous tissue kit, Qaigen) from vastus lateralis biopsy samples collected before and after (1.5 hours) a single bout of moderate intensity (75% VO2peak) cycle ergometry exercise perform by 5 unaccustomed volunteer adults (age = 24±3 yrs, BMI = 23.7±3.2). Small RNA library was prepared using the NEXTFLEX® Small RNA-Seq Kit v3. Samples were run on an SP100 flow cell with 51bp paired end reads at TGEN Collaborative Sequencing Center (Phoenix, AZ). Fastq files were analyzed using RStudio 2022.07.1+554 for Windows. All samples were trimmed and aligned to a human reference genome and genome annotation (GRCh38.14, NCBI) using the Rsubread package (Ver 2.10.5). Feature counts were obtained and differentially expressed miRNA were identified using the DESeq2 package (Ver 1.36.0). Raw counts were normalized to sequence length and depth by calculating the reads per kilobase per million to identify uniquely expressed miRNA. Gene Ontology from the DAVID online tool was utilized to explore biological processes associated with the differentially expressed miRNAs. Further analysis of predicted gene targets was conducted using the miRDB.org tool. After alignment, there was an average of 46.5 million alignments across all samples and 23.9 million successful alignments resulting in an average of 51.2% successfully aligned reads to the reference genome. An average of 34 million features were counted across all samples with an average of 3 million counts for all known miRNAs. Differential expression analysis revealed 29 miRNAs were upregulated (log 2 fold change >2, Padj <0.05) following a single bout of moderate aerobic exercise. There were no miRNA significantly down regulated. Among the 29 miRNA, 2 (miRNA 483, l2fc = 2.7±0.68, Padj = 0.14 & miRNA 3909, l2fc = Padj = 0.01) were identified in key biological processes linked to negative regulation of fat cell differentiation, inflammatory response, and blood vessel endothelial cell proliferation involved angiogenesis. One differentially expressed miRNA (miRNA 346 Padj = 0.03) was identified as a positive regulator of canonical Wnt signaling pathway involved in osteoblast differentiation. In sum, we identified 3 key miRNAs in skeletal muscle involved in regulating key biological processes in response to an acute bout of moderate intensity aerobic exercise. Future research should expand these findings by investigating interactions between identified miRNAs and target mRNA(s), and explore the existence of comparable responses in humans with known exercise response deficits such as obesity and/or aging. The study was supported by NIH/NIDDK grant R01DK094062 (CSK), the Mayo Clinic Metabolomics Resource Core through grant number U24DK100469 from the NIH/NIDDK and the Mayo Clinic CTSA grant UL1TR000135 from the National Center for Advancing Translational Sciences. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.