The Effect of Sleep Restriction, With or Without Exercise, on Skeletal Muscle Transcriptomic Profiles in Healthy Young Males

Abstract

Background: Inadequate sleep is associated with many detrimental health effects, including increased risk of developing insulin resistance and type 2 diabetes. These effects have been associated with changes to the skeletal muscle transcriptome, although this has not been characterised in response to a period of sleep restriction. Exercise induces a beneficial transcriptional response within skeletal muscle that may counteract some of the negative effects associated with sleep restriction. We hypothesised that sleep restriction would down-regulate transcriptional pathways associated with glucose metabolism, but that performing exercise would mitigate these effects.

Methods: 20 healthy young males were allocated to one of three experimental groups: a Normal Sleep (NS) group (8 h time in bed per night (TIB), for five nights (11 pm - 7 am)), a Sleep Restriction (SR) group (4 h TIB, for five nights (3 am - 7 am)), and a Sleep Restriction and Exercise group (SR+EX) (4 h TIB, for five nights (3 am - 7 am) and three high-intensity interval exercise (HIIE) sessions (performed at 10 am)). RNA sequencing was performed on muscle samples collected pre- and post-intervention. Our data was then compared to skeletal muscle transcriptomic data previously reported following sleep deprivation (24 h without sleep).

Results: Gene set enrichment analysis (GSEA) indicated there was an increased enrichment of inflammatory and immune response related pathways in the SR group post-intervention. However, in the SR+EX group the direction of enrichment in these same pathways occurred in the opposite directions. Despite this, there were no significant changes at the individual gene level from pre- to post-intervention. A set of genes previously shown to be decreased with sleep deprivation was also decreased in the SR group, but increased in the SR+EX group.

Conclusion: The alterations to inflammatory and immune related pathways in skeletal muscle, following five nights of sleep restriction, provide insight regarding the transcriptional changes that underpin the detrimental effects associated with sleep loss. Performing three sessions of HIIE during sleep restriction attenuated some of these transcriptional changes. Overall, the transcriptional alterations observed with a moderate period of sleep restriction were less evident than previously reported changes following a period of sleep deprivation.

Keywords: circadian rhythm; high-intensity interval exercise (HIE); inflammation; mitochondria; skeletal muscle; sleep loss; sleep restriction; transcriptomics.

Figure 1

(A) Study protocol and analysis overview. Participants were matched into one of three experimental groups. The study consisted of two nights of baseline sleep (8 h TIB), prior to 5 nights of either 8 h TIB (NS), or 4 h TIB (SR and SR+EX). The SR+EX group performed 3 sessions of HIIE on Day 4, 5 and 6. Skeletal muscle biopsies were collected pre- and post-intervention and underwent transcriptomic analysis. (B) Multidimensional scaling (MDS) plot showing distances between each group (blue = NS, yellow = SR, red = SR+EX), with each biopsy time points (pre- (triangles) or post-intervention (circles)). (C) Heatmap of relative expression levels of transcripts identified within the study. Individuals were grouped according to their intervention and time point (pre or post). (D) Changes in expression of core circadian clock genes for each group (Log₂FC from pre- to post-intervention). Groupings include green = normal sleep [NS], yellow = sleep restriction [SR], red = sleep restriction and exercise [SR+EX], with Pre [light purple] and Post [dark purple]. NS – Normal Sleep, SR – Sleep Restriction, SR+EX – Sleep Restriction and Exercise, HIIE – High-intensity interval exercise, TIB – Time in bed.

Figure 2

Gene set enrichment analysis (GSEA). (A) Heat map representing the top 30 significantly altered gene sets from the ‘REACTOME’ gene ontology (based on MANOVA analysis using the mitch R package (46). (B, D, F) Violin plots representing the direction of expression in each group for b) Interferon alpha/beta signalling, (D) Mitochondrial translation, and (F) Complex I biogenesis. (C, E, G) heat maps for the 20 leading-edge genes for the corresponding violin plots (Log₂FC from pre- to post-intervention). Normal Sleep (NS), Sleep Restriction (SR), and Sleep Restriction and Exercise (SR+EX). * Denotes significant within group difference from pre-intervention, adjusted P<0.05.

Figure 3

Comparison of our data set with previously reported transcriptional changes following 24 hours of sleep deprivation (13). (A) Overview of analysis workflow: two gene sets were created based on 1) Up-regulated and 2) Down-regulated genes in skeletal muscle following 24 h of sleep deprivation (SD) (created gene sets labelled as SD – Up and SD – Down, respectively). The enrichment of genes comprising SD-Up and SD-Down were then assessed in the current study cohort. Normalised enrichment score (NES) of our data set compared to the (B) SD – Up and (C) SD - Down gene sets. Rank and enrichment score of the SD - Down gene set in the (D) SR group and (E) SR+EX group, as well as heatmaps of leading-edge genes. Normal Sleep (NS), Sleep Restriction (SR), Sleep Restriction and Exercise (SR+EX), morningness-eveningness questionnaire score (MEQ score), differentially expressed genes (DEGs). *Denotes significant enrichment (adjusted P value <0.05).