Piotr Patryk Gorski

• Gorski, Piotr Patryk; Turner, Daniel; Iraki, Juma; Morton, James P; Sharples, Adam & Areta, José (2023). Human skeletal muscle methylome after low-carbohydrate energy-balanced exercise. American Journal of Physiology. Endocrinology and Metabolism. ISSN 0193-1849. 324(5), p. E437–E448. doi: 10.1152/ajpendo.00029.2023. Show summary

  We aimed to investigate the human skeletal muscle (SkM) DNA methylome after exercise in low carbohydrate (CHO) energy balance (with high fat) compared with exercise in low-CHO energy deficit (with low fat) conditions. The objective to identify novel epigenetically regulated genes and pathways associated with ‘train-low sleep-low’ paradigms. The sleep-low conditions included 9 males that cycled to deplete muscle glycogen while reaching a set energy expenditure. Post-exercise, low-CHO meals (protein-matched) completely replaced (using high-fat) or only partially replaced (low-fat) the energy expended. The following morning resting baseline biopsies were taken and the participants then undertook 75 minutes of cycling exercise, with skeletal muscle biopsies collected 30 minutes and 3.5 hours post exercise. Discovery of genome-wide DNA methylation was undertaken using Illumina EPIC arrays and targeted gene expression analysis was conducted by RT-qPCR. At baseline participants under energy balance (high fat) demonstrated a predominantly hypermethylated (60%) profile across the genome compared to energy deficit-low fat conditions. However, post exercise performed in energy balance (with high fat) elicited a more prominent hypomethylation signature 30 minutes post-exercise in gene regulatory regions important for transcription (CpG islands within promoter regions) compared with exercise in energy deficit (with low fat) conditions. Such hypomethylation was enriched within pathways related to: IL6-JAK-STAT signalling, metabolic processes, p53 / cell cycle and oxidative / fatty acid metabolism. Hypomethylation within the promoter regions of genes: HDAC2, MECR, IGF2 and c13orf16 were associated with significant increases in gene expression in the post-exercise period in energy balance compared with energy deficit. Furthermore, histone deacetylase, HDAC11 was oppositely regulated at the gene expression level compared with HDAC2, where HDAC11 was hypomethylated yet increased in energy deficit compared with energy balance conditions. Overall, we identify some novel epigenetically regulated genes associated with train-low sleep-low paradigms.

• Gorski, Piotr Patryk; Raastad, Truls; Ullrich, Max; Turner, Daniel; Hallén, Jostein & Sarvari, Sebastian [Show all 8 contributors for this article] (2022). Aerobic Exercise Training Resets the Human Skeletal Muscle Methylome Ten Years after Breast Cancer Treatment and Survival. The FASEB Journal. ISSN 0892-6638. 37(1). doi: 10.1096/fj.202201510RR. Show summary

  Background: Cancer survivors suffer impairments in skeletal muscle in terms of reduced mass and function. Interestingly, human skeletal muscle possesses an epigenetic memory of earlier stimuli, such as exercise. Long-term retention of epigenetic changes in skeletal muscle following cancer survival and/or exercise training have not yet been studied. We therefore investigated genome-wide DNA methylation (methylome) in skeletal muscle following a 5-month, 3/week aerobic-training intervention in breast cancer survivors 10-14 years after diagnosis and treatment. These results were compared to breast cancer survivors who remained untrained and to age-matched controls with no history of cancer, who undertook the same training intervention. Methods: Skeletal muscle biopsies were obtained from 23 females before(pre) and after(post) the 5-month training period. InfiniumEPIC 850K DNA methylation arrays and RT-PCR for gene expression performed. Results: The breast cancer survivors displayed a significant retention of increased DNA methylation (i.e.,hypermethylation) at a larger number of differentially methylated positions (DMPs) compared with healthy age-matched controls pre-training. Training in cancer survivors led to an exaggerated number of DMPs with a hypermethylated signature occurring at non-regulatory regions compared with training in healthy age-matched controls. However, the opposite occurred in important gene regulatory regions, where training in cancer survivors elicited a considerable reduction in methylation (i.e.,hypomethylation) in 99% of the DMPs located in CpG islands within promoter regions. Importantly, training was able to reverse the hypermethylation identified in cancer survivors back towards a hypomethylated signature that was observed pre-training in healthy age-matched controls at 300 (out of 881) of these island/promoter associated CpGs. Pathway enrichment analysis identified training in cancer survivors evoked this predominantly hypomethylated signature in pathways associated with: Cell cycle, DNA replication/repair, transcription, translation, mTOR signalling and the proteosome. Differentially methylated region (DMR) analysis also identified genes: BAG1, BTG2, CHP1, KIFC1, MKL2, MTR, PEX11B, POLD2, S100A6, SNORD104 and SPG7 as hypermethylated in breast cancer survivors, with training reversing these CpG island/promoter associated DMRs towards a hypomethylated signature. Training also elicited a largely different epigenetic response in healthy individuals than that observed in cancer survivors, with very few overlapping changes. Only one gene, SIRT2, was identified as having altered methylation in cancer survivors at baseline and after training in both the cancer survivors and healthy controls. Conclusions: Human skeletal muscle muscle may retain a hypermethylated signature as long as 10-14 years after breast cancer treatment/survival. Five months of aerobic training reset the skeletal muscle methylome towards signatures identified in healthy age-matched individuals in gene regulatory regions.

• Turner, Daniel C.; Gorski, Piotr Patryk; Seaborne, Robert A.; Viggars, Mark; Murphy, Mark & Jarvis, Jonathan C. [Show all 9 contributors for this article] (2021). Mechanical loading of bioengineered skeletal muscle in vitro recapitulates gene expression signatures of resistance exercise in vivo. Journal of Cellular Physiology. ISSN 0021-9541. 236(9), p. 6534–6547. doi: 10.1002/jcp.30328. Full text in Research Archive Show summary

  Understanding the role of mechanical loading and exercise in skeletal muscle (SkM) is paramount for delineating the molecular mechanisms that govern changes in muscle mass. However, it is unknown whether loading of bioengineered SkM in vitro adequately recapitulates the molecular responses observed after resistance exercise (RE) in vivo. To address this, the transcriptional and epigenetic (DNA methylation) responses were compared after mechanical loading in bioengineered SkM in vitro and after RE in vivo. Specifically, genes known to be upregulated/hypomethylated after RE in humans were analyzed. Ninety-three percent of these genes demonstrated similar changes in gene expression post-loading in the bioengineered muscle when compared to acute RE in humans. Furthermore, similar differences in gene expression were observed between loaded bioengineered SkM and after programmed RT in rat SkM tissue. Hypomethylation occurred for only one of the genes analysed (GRIK2) post-loading in bioengineered SkM. To further validate these findings, DNA methylation and mRNA expression of known hypomethylated and upregulated genes post-acute RE in humans were also analyzed at 0.5, 3, and 24 h post-loading in bioengineered muscle. The largest changes in gene expression occurred at 3 h, whereby 82% and 91% of genes responded similarly when compared to human and rodent SkM respectively. DNA methylation of only a small proportion of genes analyzed (TRAF1, MSN, and CTTN) significantly increased post-loading in bioengineered SkM alone. Overall, mechanical loading of bioengineered SkM in vitro recapitulates the gene expression profile of human and rodent SkM after RE in vivo. Although some genes demonstrated differential DNA methylation post-loading in bioengineered SkM, such changes across the majority of genes analyzed did not closely mimic the epigenetic response to acute-RE in humans.

• Massar, Mohd Firdaus; Turner, Daniel C.; Gorski, Piotr Patryk; Seaborne, Robert A.; Strauss, Juliette A. & Shepherd, Sam O. [Show all 12 contributors for this article] (2021). The comparative methylome and transcriptome after change of direction compared to straight line running exercise in human skeletal muscle. Frontiers in Physiology. ISSN 1664-042X. 12. doi: 10.3389/fphys.2021.619447. Full text in Research Archive Show summary

  The methylome and transcriptome signature following exercise that is physiologically and metabolic relevant to sporting contexts such as team sports or health prescription scenarios (e.g. high intensity interval training/HIIT) has not been investigated. To explore this, we undertook two different sport/exercise relevant high-intensity sprint running protocols in humans using a repeated measures design of: 1) Change of direction (COD) versus; 2) straight line (ST) sprint exercise. We took skeletal muscle biopsies from the vastus lateralis 30 minutes and 24 hours post exercise followed by 850K methylation arrays and comparative analysis with recent sprint and acute aerobic exercise meta-analysis transcriptomes. Despite matched intensity (speed x distance and number of accelerations/decelerations) between COD and ST exercise, COD exercise elicited greater movement (GPS Playerload™), physiological (HR), metabolic (lactate) as well as central and peripheral (differential RPE) loading compared with ST exercise. The exercise response alone across both conditions evoked extensive alterations in the methylome immediately post and 24 hrs after exercise, particularly in MAPK, AMPK and axon guidance pathways. COD evoked a considerably greater hypomethylated signature across the genome compared with ST sprint exercise, particularly enriched in: Protein binding, MAPK, AMPK, insulin, and axon guidance pathways. A finding that was more prominent immediately post exercise. Comparative methylome analysis with sprint running transcriptomes identified considerable overlap, with 49% of the genes altered at the expression level also differentially methylated after COD exercise. After differential methylated region analysis, we discovered that VEGFA and its downstream nuclear transcription factor, NR4A1 had enriched hypomethylation within their promoter regions. VEGFA and NR4A1 were also significantly upregulated in the sprint transcriptome and meta-analysis of exercise transcriptomes. We confirmed increased mRNA expression of VEGFA, and considerably larger increases in the expression of canonical metabolic genes, PGC1-α and NR4A3, 3 hrs post COD vs. ST exercise. Overall, we demonstrate that increased physiological load via change of direction sprint exercise in human skeletal muscle evokes considerable epigenetic modifications that are associated with changes in expression of genes responsible for adaptation to exercise. These data imply that introducing changes in direction into high intensity running protocols could serve as an important modulator of a favourable epigenomic and transcriptomic landscape in response to exercise in athletes and trigger greater skeletal muscle remodelling through enhanced gene expression.

• Hughes, David C.; Turner, Daniel; Baehr, Leslie M; Seaborne, Robert A.; Viggars, Mark & Jarvis, Jonathan C. [Show all 11 contributors for this article] (2021). Knockdown of the E3 Ubiquitin ligase UBR5 and its role in skeletal muscle anabolism. American Journal of Physiology - Cell Physiology. ISSN 0363-6143. 320(1), p. C45–C56. doi: 10.1152/AJPCELL.00432.2020. Full text in Research Archive
• Turner, Daniel C.; Gorski, Piotr Patryk; Massar, Mohd Firdaus; Seaborne, Robert A.; Baumert, Phillipp & Brown, Alexander D. [Show all 22 contributors for this article] (2020). DNA methylation across the genome in aged human skeletal muscle tissue and muscle-derived cells: the role of HOX genes and physical activity. Scientific Reports. ISSN 2045-2322. 10(1). doi: 10.1038/s41598-020-72730-z. Full text in Research Archive Show summary

  Skeletal muscle tissue demonstrates global hypermethylation with age. However, methylome changes across the time-course of differentiation in aged human muscle derived cells, and larger coverage arrays in aged muscle tissue have not been undertaken. Using 850K DNA methylation arrays we compared the methylomes of young (27 ± 4.4 years) and aged (83 ± 4 years) human skeletal muscle and that of young/aged heterogenous muscle-derived human primary cells (HDMCs) over several time points of differentiation (0, 72 h, 7, 10 days). Aged muscle tissue was hypermethylated compared with young tissue, enriched for; pathways-in-cancer (including; focal adhesion, MAPK signaling, PI3K-Akt-mTOR signaling, p53 signaling, Jak-STAT signaling, TGF-beta and notch signaling), rap1-signaling, axon-guidance and hippo-signalling. Aged cells also demonstrated a hypermethylated profile in pathways; axon-guidance, adherens-junction and calcium-signaling, particularly at later timepoints of myotube formation, corresponding with reduced morphological differentiation and reductions in MyoD/Myogenin gene expression compared with young cells. While young cells showed little alterations in DNA methylation during differentiation, aged cells demonstrated extensive and significantly altered DNA methylation, particularly at 7 days of differentiation and most notably in focal adhesion and PI3K-AKT signalling pathways. While the methylomes were vastly different between muscle tissue and HDMCs, we identified a small number of CpG sites showing a hypermethylated state with age, in both muscle tissue and cells on genes KIF15, DYRK2, FHL2, MRPS33, ABCA17P. Most notably, differential methylation analysis of chromosomal regions identified three locations containing enrichment of 6–8 CpGs in the HOX family of genes altered with age. With HOXD10, HOXD9, HOXD8, HOXA3, HOXC9, HOXB1, HOXB3, HOXC-AS2 and HOXC10 all hypermethylated in aged tissue. In aged cells the same HOX genes (and additionally HOXC-AS3) displayed the most variable methylation at 7 days of differentiation versus young cells, with HOXD8, HOXC9, HOXB1 and HOXC-AS3 hypermethylated and HOXC10 and HOXC-AS2 hypomethylated. We also determined that there was an inverse relationship between DNA methylation and gene expression for HOXB1, HOXA3 and HOXC-AS3. Finally, increased physical activity in young adults was associated with oppositely regulating HOXB1 and HOXA3 methylation compared with age. Overall, we demonstrate that a considerable number of HOX genes are differentially epigenetically regulated in aged human skeletal muscle and HDMCs and increased physical activity may help prevent age-related epigenetic changes in these HOX genes.

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• Sharples, Adam; Turner, Daniel; Roth, Stephen; Seaborne, Robert A.; Egan, Brendan & Viggars, Mark [Show all 11 contributors for this article] (2022). Methods in molecular exercise physiology. In Sharples, Adam; Morton, James P & Wackerhage, Henning (Ed.), Molecular Exercise Physiology: An Introduction (Second Edition) . Routledge. ISSN 9781315110752. p. 19–60. doi: 10.4324/9781315110752-2. Show summary

  In this chapter, we describe and explain some of the most important methodology that molecular exercise physiologists use, at level of the molecule and cell, to investigate the microscopic make-up of our cell’s response to exercise. We discuss the important role of blood and muscle biopsy sampling and describe and explain, in detail, the different types of analysis that might be undertaken by molecular exercise physiologists at the molecular (DNA, RNA, protein), cellular and tissue levels. From a muscle tissue biopsy, we describe the process of isolating DNA, RNA, protein and processing of tissue/cells for immunolabelling analysis, as well as primary muscle-derived cell isolation for growing muscle cells in a dish (in-vitro culture). We describe and explain the methods used to analyse a person’s inherited genetic code and sequence variation of DNA between individuals at both the single/targeted gene level and across the genome (genomics), and how to investigate epigenetic modifications to DNA and chromatin again, at both the individual gene level and across the genome (epigenomics). We describe and explain the methods for determining whether our genes are turned on or off in response to exercise at the single gene level and across the ‘transcriptome’ (transcriptomics). We describe and explain the methods for analyses of protein, to measure the abundance and activity of proteins and their role in ‘signal transduction’ in response to exercise at the individual protein level and across the ‘proteome’ (proteomics). We also include a description of the most important immunolabelling methods assessing changes at the protein level in skeletal muscle cell/tissue level that may help confirm the changes (or cellular location of those changes) observed at the molecular level. Finally, we will provide a narrative for the technique of primary muscle cell isolation from human biopsies, and how these isolated cells can be used as ‘in a dish’ (in-vitro) culture models to mimic exercise (in both monolayer and three-dimensional cultures) to compliment molecular analyses from whole-body (in-vivo) human exercise research.

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