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Scientific Programme

Physiology & Nutrition

OP-PN04 - Molecular Biology and Biochemistry I

Date: 03.07.2024, Time: 09:30 - 10:45, Lecture room: Alsh 1

Description

Chair TBA

Chair

TBA
TBA
TBA

ECSS Paris 2023: OP-PN04

Speaker A Dale Taylor

Speaker A

Dale Taylor
Victoria University, Institute for Health and Sport
Australia
"New insights into exercise-induced skeletal muscle gene expression in men and women"

INTRODUCTION: Exercise stimulates numerous adaptations in skeletal muscle, including increased mitochondrial content. The currently accepted dogma proposes that transient changes in the mRNA of mitochondrial genes after a single session of exercise are a major determinant of subsequent adaptations to training. However, the use of only 1 or 2 time-points post exercise, small sample sizes, and a lack of consideration for the effect of sex, has left major gaps in our understanding of exercise-induced mitochondrial gene expression. METHODS: RNA sequencing was performed on muscle biopsies collected before, during, and 0, 3, 6, 9, 12, 24, and 48 hours following a single session of high-intensity interval exercise from 20 healthy untrained men (27.3 ± 6.3 y; 179.6 ± 10.2 cm; 81.5 ± 12.5 kg) and 20 healthy untrained women (27.6 ± 5.6 y; 166.5 ± 7.8 cm; 68.0 ± 9.9 kg). The exercise session consisted of 4 x 4-min intervals at an intensity between each participant’s lactate threshold (LT) and peak power output (PPO), interspersed with 2 min of recovery. RESULTS: Our adoption of the most extensive post-exercise biopsy time course to date allowed us to identify more than 10,000 genes never previously reported to be differentially expressed by aerobic exercise; the vast majority were identified 9 to 48 hours post-exercise. Of these, 1016 were mitochondrial genes, with over 800 not previously reported to be altered by aerobic exercise. Soft clustering of the 1,016 mitochondrial differentially expressed genes revealed six characteristic patterns of expression, including clusters of early (3-6 h), mid (9-12 h), and late (24-48 h) responding genes. We also identified 675 mitochondrial genes that were downregulated 24-48 h post exercise, including most oxidative phosphorylation complex subunits. Transcription factor enrichment analysis using an aggregated ChIP-Seq dataset library identified the MYC transcription factor network as a key regulator for many of these gene clusters. Similarly, p53 was identified as an important regulator of late-responding genes. Although there were over 500 genes detected as differentially expressed between men and women at baseline, sex had a minimal effect on the expression of mitochondrial genes in response to exercise. CONCLUSION: Our unique study was able to produce the most extensive description to date of the exercise-induced transcriptome, with over 14,000 genes identified as differentially expressed. Interestingly, several diverging patterns of expression for mitochondrial genes were found, indicating regulation by various transcriptional factor networks and potentially a high degree of variance in post-transcriptional regulation. By obtaining a statistical power above 0.85, and use of an exercise intensity based on both LT and PPO, we were able to detect fewer sex differences for exercise-induced mitochondrial gene expression than previously assumed.

Read CV Dale Taylor

ECSS Paris 2023: OP-PN04

Speaker B Connor Stead

Speaker B

Connor Stead
Liverpool John Moores University, Research Institute of Sport and Exercise Science
United Kingdom
"Dynamic proteomic responses to aerobic versus resistance training in human skeletal muscle."

INTRODUCTION: We investigated muscle adaptations to aerobic training (AT) versus resistance training (RT) during the early untrained-state versus longer-term trained-state using novel deuterium oxide labelling and proteomic techniques. Our study aims to generate new insight into training mode-specific muscle adaptations by measuring protein-specific changes in abundance and synthesis rates. METHODS: In a within-subject longitudinal design, 14 (8F/6M) healthy individuals (20± 2y, body mass: 70±21 Kg) completed 10-wks of thrice weekly unilateral resistance (RT: 3 sets × 10-12 reps 80% 1-repetition maximum (RM) leg press and leg extension) and unilateral aerobic (AT: 4 × 5 min one-legged cycling at 65% Wattmax) training. Biopsies were taken at the start and end of a 1-week free-living period prior to training (Baseline), the 1st-wk of training (Early), and the 10th-wk of training (Later). Participants consumed deuterium oxide across all 3 study periods. Muscle samples (n = 5 individuals, 1M/4F) were analysed by liquid chromatography tandem-mass spectrometry. Within-subject 2-way ANOVA investigated interactions between exercise mode (RT vs AT) and study period (Baseline x Early x Later) in protein-specific abundance and synthesis rates. Significant differences (p<0.05) were investigated using bioinformatic analyses (proteins reported as UniProt identifiers). RESULTS: RT increased (p<0.005) 1RM leg press 116% (+65±10kg) and 1RM leg extension 76% (+16±5kg), whereas AT increased (p=0.025) unilateral Wmax 17% (+21±17W). Proteomic analysis quantified 2883 abundance and 1465 synthesis rates. Mixed-protein FSR (%/d) increased (34% NS) during Early RT only and was 15% above Baseline during Later RT and AT. Early RT increased the turnover of 22 (primarily myofibrillar) proteins, whereas Early AT increased the turnover of glycolytic enzymes and the abundances of 10 proteins of the KEGG pathway ‘Oxidative phosphorylation’ (OXPHOS; FDR = 0.03). Later AT increased the abundance of proteins associated with ‘Cellular respiration’ (FDR = 1.2e-17), including 16 Complex I (CI) subunits. Both RT and AT significantly increased abundance of 48 mitochondrial proteins, including 5 CI accessory subunits and regulators of mitochondrial quality (OPA1, MAIP1, AFG32, CHC10, MIC26, and GHITM). Later AT specifically increased the turnover of regulators of mitochondrial proteostasis, including the HSP70 co-chaperone BAG3 and eIF5A. Later RT specifically increased the turnover of 21 proteins, including glycolytic enzymes and regulators of mitochondrial morphology and quality control, such as CHCHD2 which increased (p < 0.01) in turnover rate from 5.7±4.9 %/d at Baseline to 13.6±2.8 %/d in Later RT. CONCLUSION: AT and RT resulted in improvements in mitochondrial quality via training-mode specific changes in abundance and synthesis rates. Remodelling of CI was specific to AT, whereas RT increased in the abundance and turnover of proteins associated with mitochondrial quality independent of changes in OXPHOS catalytic subunits.

Read CV Connor Stead

ECSS Paris 2023: OP-PN04

Speaker C Maria Hansen

Speaker C

Maria Hansen
University of Copenhagen, Department of Biomedical Sciences
Denmark
"Single-nuclei gene expression in skeletal muscle does not mirror a training-induced increase in insulin sensitivity in individuals with type 2 diabetes"

INTRODUCTION: Training improves insulin sensitivity in patients with type 2 diabetes (T2D) (1), but the underlying mechanisms are only partly understood. Single-nuclei RNA-sequencing (snRNA-seq) offers the opportunity to study fibre type-specific gene expression changes. This is the first study to examine nuclei-specific gene expression changes in skeletal muscle in individuals with T2D and healthy controls (CON) in response to a short-term high-intensity training (HIIT) program. We hypothesised that snRNA-seq of muscle biopsies would reveal marked diabetes- and training-induced responses. METHODS: Ten males with T2D (57±2 years, BMI 31±1 kg/m2, HbA1c 53±1 mmol/mol) and ten male CON (53±2 years, BMI 31±1 kg/m2, HbA1c 37±1 mmol/mol) completed two weeks of one-legged HIIT on a cycle ergometer. Insulin sensitivity (clamp + leg balance technique) was measured previously (2). We prepared a single nuclei suspension from muscle biopsies from untrained (UT) and trained (T) legs and used the 10X Genomics system for the preparation of snRNA-seq Libraries, which were sequenced using an Illumina NextSeq550snRNA-seq. QC, cluster identification and differential expression testing were analysed using the Seurat (v4), and we identified differentially expressed genes (DEGs) between two groups of nuclei using a Wilcoxon Rank Sum test. Fibre type distribution was confirmed through immunohistochemistry. RESULTS: Insulin-stimulated leg glucose clearance was 33±24% higher in T legs compared to UT legs in both groups (2). Single-nuclei RNA-sequencing in 38 biopsies yielded 135,225 nuclei in total, and we profiled 99,970 myonuclei. In the UT legs, there were 249 DEGs between the groups, with equal distribution between fibre types. When comparing UT and T legs, there were no differences between the number of DEGs in the groups (222 DEGs in CON and 234 DEGs in T2D). Almost no genes related to the citric acid cycle, glycolysis, glycogenolysis, or beta-oxidation differed between the groups in the UT leg. In T2D there was no effect of training on genes related to glycolysis and glycogenolysis. However, CON had multiple DEGs for glycolytic and glycogenolytic enzymes (i.e. enolase, glycogen debranching enzyme, glycogen phosphorylase) in the type 2A and type 2X fibres in the T leg compared to the UT leg. There were no changes in fibre type distribution after training, and the immunostaining and the snRNA-seq fibre type distributions were in accordance with each other. CONCLUSION: HIIT improves skeletal muscle insulin sensitivity, but the modest single-nuclei gene expression changes did not mirror this response. Mostly, genes related to glycolysis and glycogenolysis in type 2 muscle fibres displayed significant differences between the groups in response to training. Overall, the pattern of altered gene expressions does not seem to explain the marked change in muscle insulin sensitivity. References 1. Kanaley et al., Med Sci Sports Exerc, 2022 2. Dela F et al., Acta Physiol, 2019

Read CV Maria Hansen

ECSS Paris 2023: OP-PN04