Murine myoblast migration: influence of replicative ageing and nutrition

Cell migration is central to skeletal muscle repair following damage. Leucine and β-Hydroxy β-methylbutyric acid (HMB) are supplements consumed for recovery from muscle damaging exercise in humans, however, their impact on muscle cell migration with age is not yet understood. We hypothesised that replicatively aged (“aged”; P46–P48) myoblasts would be less efficient at basal and supplemented repair versus parental controls (“control”; P12–P16). Aged and control myoblasts were scratch-damaged and migration velocity, directionality and distance assessed over 48 h in the absence and presence of leucine (10 mM) or HMB (10 mM) ± PI3K/Akt (LY294002 10 μM), ERK (PD98059 5 μM) or mTOR (rapamycin 0.5 μM) inhibition. Opposing our hypothesis, aged cells displayed increased velocities, directionality and distance migrated (P < 0.001) versus control. Leucine and HMB significantly increased (P < 0.001) the same parameters in control cells. The supplements were with smaller, albeit significant impact on aged cell velocity (P < 0.001) and in the presence of HMB only, distance (P = 0.041). Inhibitor studies revealed that, PI3K and ERK activation were essential for velocity, directionality and migration distance of aged cells in basal conditions, whereas mTOR was important for directionality only. While PI3K activation was critical for all parameters in control cells (P < 0.001), inhibition of ERK or mTOR improved, rather than reduced, control cell migration distance. Enhanced basal velocity, directionality and distance in aged cells required ERK and PI3K activation. By contrast, in control cells, basal migration was underpinned by PI3K activation, and facilitated by leucine or HMB supplementation, to migration levels seen in aged cells. These data suggest that replicatively aged myoblasts are not anabolically resistant per se, but are capable of efficient repair, underpinned by altered signaling pathways, compared with unaged control myoblasts

Does skeletal muscle have an ‘epi’-memory? The role of epigenetics in nutritional programming, metabolic disease, aging and exercise

Skeletal muscle mass, quality and adaptability are fundamental in promoting muscle performance, maintaining metabolic function and supporting longevity and healthspan. Skeletal muscle is programmable and can ‘remember’ early-life metabolic stimuli affecting its function in adult life. In this review, the authors pose the question as to whether skeletal muscle has an ‘epi’-memory? Following an initial encounter with an environmental stimulus, we discuss the underlying molecular and epigenetic mechanisms enabling skeletal muscle to adapt, should it re-encounter the stimulus in later life. We also define skeletal muscle memory and outline the scientific literature contributing to this field. Furthermore, we review the evidence for early-life nutrient stress and low birth weight in animals and human cohort studies, respectively, and discuss the underlying molecular mechanisms culminating in skeletal muscle dysfunction, metabolic disease and loss of skeletal muscle mass across the lifespan. We also summarize and discuss studies that isolate muscle stem cells from different environmental niches in vivo (physically active, diabetic, cachectic, aged) and how they reportedly remember this environment once isolated in vitro. Finally, we will outline the molecular and epigenetic mechanisms underlying skeletal muscle memory and review the epigenetic regulation of exercise-induced skeletal muscle adaptation, highlighting exercise interventions as suitable models to investigate skeletal muscle memory in humans. We believe that understanding the ‘epi’-memory of skeletal muscle will enable the next generation of targeted therapies to promote muscle growth and reduce muscle loss to enable healthy aging

Mimicking exercise in three-dimensional bioengineered skeletal muscle to investigate cellular and molecular mechanisms of physiological adaptation

Bioengineering of skeletal muscle in vitro in order to produce highly aligned myofibres in relevant three dimensional (3D) matrices have allowed scientists to model the in vivo skeletal muscle niche. This review discusses essential experimental considerations for developing bioengineered muscle in order to investigate exercise mimicking stimuli. We identify current knowledge for the use of electrical stimulation and co-culture with motor neurons to enhance skeletal muscle maturation and contractile function in bioengineered systems in vitro. Importantly, we provide a current opinion on the use of acute and chronic exercise mimicking stimuli (electrical stimulation and mechanical overload) and the subsequent mechanisms underlying physiological adaptation in 3D bioengineered muscle. We also identify that future studies using the latest bioreactor technology, providing simultaneous electrical and mechanical loading and flow perfusion in vitro, may provide the basis for advancing knowledge in the future. We also envisage, that more studies using genetic, pharmacological, and hormonal modifications applied in human 3D bioengineered skeletal muscle may allow for an enhanced discovery of the in-depth mechanisms underlying the response to exercise in relevant human testing systems. Finally, 3D bioengineered skeletal muscle may provide an opportunity to be used as a pre-clinical in vitro test-bed to investigate the mechanisms underlying catabolic disease, while modelling disease itself via the use of cells derived from human patients without exposing animals or humans (in phase I trials) to the side effects of potential therapies

Skeletal muscle cells possess a 'memory' of acute early life TNF-α exposure: role of epigenetic adaptation.

Sufficient quantity and quality of skeletal muscle is required to maintain lifespan and healthspan into older age. The concept of skeletal muscle programming/memory has been suggested to contribute to accelerated muscle decline in the elderly in association with early life stress such as fetal malnutrition. Further, muscle cells in vitro appear to remember the in vivo environments from which they are derived (e.g. cancer, obesity, type II diabetes, physical inactivity and nutrient restriction). Tumour-necrosis factor alpha (TNF-α) is a pleiotropic cytokine that is chronically elevated in sarcopenia and cancer cachexia. Higher TNF-α levels are strongly correlated with muscle loss, reduced strength and therefore morbidity and earlier mortality. We have extensively shown that TNF-α impairs regenerative capacity in mouse and human muscle derived stem cells [Meadows et al. (J Cell Physiol 183(3):330-337, 2000); Foulstone et al. (J Cell Physiol 189(2):207-215, 2001); Foulstone et al. (Exp Cell Res 294(1):223-235, 2004); Stewart et al. (J Cell Physiol 198(2):237-247, 2004); Al-Shanti et al. (Growth factors (Chur, Switzerland) 26(2):61-73, 2008); Saini et al. (Growth factors (Chur, Switzerland) 26(5):239-253, 2008); Sharples et al. (J Cell Physiol 225(1):240-250, 2010)]. We have also recently established an epigenetically mediated mechanism (SIRT1-histone deacetylase) regulating survival of myoblasts in the presence of TNF-α [Saini et al. (Exp Physiol 97(3):400-418, 2012)]. We therefore wished to extend this work in relation to muscle memory of catabolic stimuli and the potential underlying epigenetic modulation of muscle loss. To enable this aim; C2C12 myoblasts were cultured in the absence or presence of early TNF-α (early proliferative lifespan) followed by 30 population doublings in the absence of TNF-α, prior to the induction of differentiation in low serum media (LSM) in the absence or presence of late TNF-α (late proliferative lifespan). The cells that received an early plus late lifespan dose of TNF-α exhibited reduced morphological (myotube number) and biochemical (creatine kinase activity) differentiation vs. control cells that underwent the same number of proliferative divisions but only a later life encounter with TNF-α. This suggested that muscle cells had a morphological memory of the acute early lifespan TNF-α encounter. Importantly, methylation of myoD CpG islands were increased in the early TNF-α cells, 30 population doublings later, suggesting that even after an acute encounter with TNF-α, the cells have the capability of retaining elevated methylation for at least 30 cellular divisions. Despite these fascinating findings, there were no further increases in myoD methylation or changes in its gene expression when these cells were exposed to a later TNF-α dose suggesting that this was not directly responsible for the decline in differentiation observed. In conclusion, data suggest that elevated myoD methylation is retained throughout muscle cells proliferative lifespan as result of early life TNF-α treatment and has implications for the epigenetic control of muscle loss

Impaired hypertrophy in myoblasts is improved with testosterone administration

We investigated the ability of testosterone (T) to restore differentiation in multiple population doubled (PD) murine myoblasts, previously shown to have reduced differentiation in monolayer and bioengineered skeletal muscle cultures vs. their parental controls (CON) (Sharples et al., 2011, 2012 [7] and [26]). Cells were exposed to low serum conditions in the presence or absence of T (100 nM) ± PI3K inhibitor (LY294002) for 72 h and 7 days (early and late muscle differentiation respectively). Morphological analyses were performed to determine myotube number, diameter (μm) and myonuclear accretion as indices of differentiation and myotube hypertrophy. Changes in gene expression for myogenin, mTOR and myostatin were also performed. Myotube diameter in CON and PD cells increased from 17.32 ± 2.56 μm to 21.02 ± 1.89 μm and 14.58 ± 2.66 μm to 18.29 ± 3.08 μm (P ≤ 0.05) respectively after 72 h of T exposure. The increase was comparable in both PD (+25%) and CON cells (+21%) suggesting a similar intrinsic ability to respond to exogenous T administration. T treatment also significantly increased myonuclear accretion (% of myotubes expressing 5+ nuclei) in both cell types after 7 days exposure (P ≤ 0.05). Addition of PI3K inhibitor (LY294002) in the presence of T attenuated these effects in myotube morphology (in both cell types) suggesting a role for the PI3K pathway in T stimulated hypertrophy. Finally, PD myoblasts showed reduced responsiveness to T stimulated mRNA expression of mTOR vs. CON cells and T also reduced myostatin expression in PD myoblasts only. The present study demonstrates testosterone administration improves hypertrophy in myoblasts that basally display impaired differentiation and hypertrophic capacity vs. their parental controls, the action of testosterone in this model was mediated by PI3K/Akt pathway

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