Mimicking exercise in vitro: effects of myotube contractions and mechanical stretch on omics.

The number of studies using skeletal muscle (SkM) cell culture models to study exercise in vitro are rapidly expanding. Progressively, more comprehensive analysis methods, such as different omics approaches including transcriptomics, proteomics, and metabolomics have been used to examine the intra- and extracellular molecular responses to exercise mimicking stimuli in cultured myotubes. Among other techniques, exercise-like electrical pulse stimulation (EL-EPS) and mechanical stretch of SkM cells are the two most commonly used methods to mimic exercise in vitro. In this mini-review, we focus on these two approaches and their effects on the omics of myotubes and/or cell culture media. Furthermore, besides traditional two-dimensional (2-D) methods, the use of three-dimensional (3-D) SkM approaches are increasing in the field of in vitro exercise mimicry. Our aim with this mini-review is to provide the reader with an up-to-date overview of the 2-D and 3-D models and the use of omics approaches to study the molecular response to exercise in vitro.

DNA methylation across the genome in aged human skeletal muscle tissue and muscle-derived cells: the role of HOX genes and physical activity.

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.

PGC-1α alternative promoter (Exon 1b) controls augmentation of total PGC-1α gene expression in response to cold water immersion and low glycogen availability.

This investigation sought to determine whether post-exercise cold water immersion and low glycogen availability, separately and in combination, would preferentially activate either the Exon 1a or Exon 1b Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) promoter. Through a reanalysis of sample design, we identified that the systemic cold-induced augmentation of total PGC-1α gene expression observed previously (Allan et al. in J Appl Physiol 123(2):451-459, 2017) was largely a result of increased expression from the alternative promoter (Exon 1b), rather than canonical promoter (Exon 1a). Low glycogen availability in combination with local cooling of the muscle (Allan et al. in Physiol Rep 7(11):e14082, 2019) demonstrated that PGC-1α alternative promoter (Exon 1b) expression continued to rise at 3 h post-exercise in all conditions; whilst, expression from the canonical promoter (Exon 1a) decreased between the same time points (post-exercise-3 h post-exercise). Importantly, this increase in PGC-1α Exon 1b expression was reduced compared to the response of low glycogen or cold water immersion alone, suggesting that the combination of prior low glycogen and CWI post-exercise impaired the response in gene expression versus these conditions individually. Data herein emphasise the influence of post-exercise cooling and low glycogen availability on Exon-specific control of total PGC-1 α gene expression and highlight the need for future research to assess Exon-specific regulation of PGC-1α.

Methylome of human skeletal muscle after acute & chronic resistance exercise training, detraining & retraining.

DNA methylation is an important epigenetic modification that can regulate gene expression following environmental encounters without changes to the genetic code. Using Infinium MethylationEPIC BeadChip Arrays (850,000 CpG sites) we analysed for the first time, DNA isolated from untrained human skeletal muscle biopsies (vastus lateralis) at baseline (rest) and immediately following an acute (single) bout of resistance exercise. In the same participants, we also analysed the methylome following a period of muscle growth (hypertrophy) evoked via chronic (repeated bouts-3 sessions/wk) resistance exercise (RE) (training) over 7-weeks, followed by complete exercise cessation for 7-weeks returning muscle back to baseline levels (detraining), and finally followed by a subsequent 7-week period of RE-induced hypertrophy (retraining). These valuable methylome data sets described in the present manuscript and deposited in an open-access repository can now be shared and re-used to enable the identification of epigenetically regulated genes/networks that are modified after acute anabolic stimuli and hypertrophy, and further investigate the phenomenon of epigenetic memory in skeletal muscle.

Acute mechanical overload increases IGF-I and MMP-9 mRNA in 3D tissue-engineered skeletal muscle.

Skeletal muscle (SkM) is a tissue that responds to mechanical load following both physiological (exercise) or pathophysiological (bed rest) conditions. The heterogeneity of human samples and the experimental and ethical limitations of animal studies provide a rationale for the study of SkM plasticity in vitro. Many current in vitro approaches of mechanical loading of SkM disregard the three-dimensional (3D) structure in vivo. Tissue engineered 3D SkM, that displays highly aligned and differentiated myotubes, was used to investigate mechano-regulated gene transcription of genes implicated in hypertrophy/atrophy. Static loading (STL) and ramp loading (RPL) at 10 % strain for 60 min were used as mechano-stimulation with constructs sampled immediately for RNA extraction. STL increased IGF-I mRNA compared to both RPL and CON (control, p = 0.003 and 0.011 respectively) whilst MMP-9 mRNA increased in STL and RPL compared to CON (both p < 0.05). IGFBP-2 mRNA was differentially regulated in RPL and STL compared to CON (p = 0.057), whilst a reduction in IGFBP-5 mRNA was found for STL and RPL compared to CON (both p < 0.05). There was no effect in the expression of putative atrophic genes, myostatin, MuRF-1 and MAFBx (all p > 0.05). These data demonstrate a transcriptional signature associated with SkM hypertrophy within a tissue-engineered model that more greatly recapitulates the in vivo SkM structure compared previously published studies.