PLoS Genet. 2016 Sep 22;12(9):e1006294. doi: 10.1371/journal.pgen.1006294.

The Impact of Endurance Training on Human Skeletal Muscle Memory, Global Isoform Expression and Novel Transcripts.




Regular physical activity is an environmental stimulus that is highly associated to many health benefits, while physical inactivity is detrimental for health. Regular exercise training is used in the prevention and treatment of a large number of disease conditions, and reduces the risk for premature death (1). Exercise-induced adaptation of skeletal muscle is important for muscle function as well as the whole-body health effects of training. The understanding of the regulation of gene expression changes in response to training has progressed extensively over the past 20 years. Still, many key mechanisms remain to be investigated. This study investigated the skeletal muscle transcriptome changes, with specific focus on isoforms, in response to repeated periods of endurance training and the potential presence of a residual intrinsic memory of previous endurance training that could influence the response to a repeated training period after detraining.


The study was designed as a controlled, repeated intervention study where each individual was their own control. Twenty-three healthy volunteers performed supervised one-legged knee-extension exercise training four times per week for twelve weeks, in total 45 sessions. In the first training period, all subjects exercised only one randomized leg. After nine months of detraining, a subset of the subjects returned to perform a second training period, now training both legs (2×45 min each session, four times/week). The study design is outlined in Figure 1A. Two biopsies from the vastus lateralis muscle of each leg were obtained at rest before and after each training period (Figure 1B).


Multivariate statistics was applied to RNA-seq data, showing that three months of endurance training induced significant transcriptional changes in human skeletal muscle. 2394 genes were differentially expressed in the trained leg. At the isoform level, 3404 isoforms, distributed across 2624 genes, were differentially expressed. For a majority of the genes, only one isoform was thus differentially expressed, while 2 % of the genes differentially expressed 4-8 isoforms. The NDRG2 gene, with 18 differentially expressed isoforms, displayed 17 isoforms decreasing and one isoform increasing in expression. Gene ontology analysis showed that the differentially expressed isoforms were mainly associated to cellular respiration and ATP synthesis coupled electron transport, and enriched pathways included oxidative phosphorylation and the TCA cycle. Fifty-four genes differentially expressed multiple isoforms that changed in opposite directions with training. This information shows the complexity of gene regulation and the importance of recognizing this before drawing conclusions solely from gene-based data. Examples of these genes include the mitochondrial ATP synthase ATP5G1, the FUS gene (Fused In Sarcoma) that encodes an RNA-binding protein involved in pre-mRNA processing, and Pyruvate Kinase Muscle (PKM). A potential reason for differential regulation of isoforms from the same gene is that the isoforms have different functions and therefore contrasting roles in muscle adaptation to training.


In order to investigate if endurance training could alter expression of transcripts in unannotated parts of the genome, we performed a differential expression analysis of 2400 novel transcripts identified in skeletal muscle in a previous paper from our group (2). We found 34 transcripts that were differentially expressed and not annotated in Ensembl v.71, nine were upregulated and 25 were downregulated with training. All transcripts contained predicted open reading frames (ORFs) and protein-coding motifs, suggesting that they have protein-coding potential. A majority of the assembled transcripts also overlapped conserved regions in chimp, rhesus, mouse, rat or dog, indicating that they are functionally interesting. Two transcripts were also identified as enhancer RNAs when compared to a paper by Arner et al. (3).


The repeated training period after detraining was subsequently used to study a potential memory at the transcriptional level. Before the start of Period 2, there were no significant remaining effects from the previous training period in the previously trained leg. Furthermore, upon retraining, the transcriptomic response was largely consistent with the first training period. Therefore, the data indicated no detectable muscle memory at the transcriptome level, which could explain why it is hard to get fit again after a physically inactive period.



Figure 1. Study design. A) The outline of the study, with performance tests, biopsy time points with the corresponding code (T (for the leg trained in Period 1) or U (for the untrained leg in Period 1) followed by the time point (1-4)). B) Illustration of the biopsy sites in vastus lateralis. C) Schematic image of the one-legged training regime.



1. F. W. Booth, C. K. Roberts, M. J. Laye, Lack of exercise is a major cause of chronic diseases. Compr Physiol 2, 1143-1211 (2012).
2. M. E. Lindholm et al., The human skeletal muscle transcriptome: sex differences, alternative splicing, and tissue homogeneity assessed with RNA sequencing. Faseb J 28, 4571-4581 (2014).
3. E. Arner et al., Gene regulation. Transcribed enhancers lead waves of coordinated transcription in transitioning mammalian cells. Science 347, 1010-1014 (2015).