Adam P. Sharples

Longevity and skeletal muscle mass: the role of IGF signalling, the sirtuins, dietary restriction and protein intake.

Advancing age is associated with a progressive loss of skeletal muscle (SkM) mass and function. Given the worldwide aging demographics, this is a major contributor to morbidity, escalating socio‐economic costs and ultimately mortality. Previously, it has been established that a decrease in regenerative capacity in addition to SkM loss with age coincides with suppression of insulin/insulin‐like growth factor signalling pathways. However, genetic or pharmacological modulations of these highly conserved pathways have been observed to significantly enhance life and healthspan in various species, including mammals. This therefore provides a controversial paradigm in which reduced regenerative capacity of skeletal muscle tissue with age potentially promotes longevity of the organism. This paradox will be assessed and considered in the light of the following: (i) the genetic knockout, overexpression and pharmacological models that induce lifespan extension (e.g. IRS‐1/s6K KO, mTOR inhibition) versus the important role of these signalling pathways in SkM growth and adaptation; (ii) the role of the sirtuins (SIRTs) in longevity versus their emerging role in SkM regeneration and survival under catabolic stress; (iii) the role of dietary restriction and its impact on longevity versus skeletal muscle mass regulation; (iv) the crosstalk between cellular energy metabolism (AMPK/TSC2/SIRT1) and survival (FOXO) versus growth and repair of SkM (e.g. AMPK vs. mTOR); and (v) the impact of protein feeding in combination with dietary restriction will be discussed as a potential intervention to maintain SkM mass while increasing longevity and enabling healthy aging.

C2 and C2C12 murine skeletal myoblast models of atrophic and hypertrophic potential: relevance to disease and ageing?

Reduced muscle mass and increased susceptibility to TNF‐induced degradation accompany inflamed ageing and chronic diseases. Furthermore, C2 myoblasts display diminished differentiation and increased susceptibility to TNF‐α‐induced cell death versus subcloned C2C12 cells, providing relevant models to assess: differentiation (creatine kinase), growth (protein), death (trypan‐blue) and anabolic/catabolic parameters (RT‐PCR) over 72 h ± TNF‐α (20 ng ml−1). At 48 and 72 h, respectively, larger myotubes and significantly higher CK activity (320.26 ± 6.82 vs. 30.71 ± 2.5, P < 0.05; 544.94 ± 27.7 vs. 39.4 ± 3.37 mU mg ml−1, P < 0.05), fold increases in myoD (21.45 ± 3.12 vs. 3.97 ± 1.76, P < 0.05; 31.07 ± 3.1 vs. 6.82 ± 1.93, P < 0.05) and myogenin mRNA (241.8 ± 40 vs. 36.80 ± 19.3, P < 0.05; 440 ± 100.5 vs. 201.1 ± 86, P < 0.05) were detected in C2C12 versus C2. C2C12 showed significant increases in IGF‐I mRNA (243.05 ± 3.87 vs. 105.75 ± 21.95, P < 0.05), reduced proliferation and significantly lower protein expression (1.21 ± 0.28 vs. 1.79 ± 0.29 mg ml−1, P < 0.05) at 72 h versus C2 cells. Significant temporal reductions in C2C12 IGFBP2 mRNA (28.02 ± 15.44, 13.82 ± 8.07, 6.92 ± 4.37, P < 0.05) contrasted increases in C2s (4.31 ± 3.31, 13.02 ± 9.92, 82.9 ± 58.9, P < 0.05) at 0, 48 and 72 h, respectively. TNF‐α increased cell death in C2s (2.67 ± 1.54%, 34.42 ± 5.39%, 29.71 ± 5.79% (0, 48, 72 h), P < 0.05), yet was without effect in C2C12s at 48 h but caused a small significant increase at 72 h (9.88 ± 4.02% (TNF‐α) vs. 6.17 ± 0.749% (DM), 72 h). TNF‐α and TNFRI mRNA were unchanged; however, larger reductions in IGF‐I (8.2‐ and 7.5‐fold vs. 4.5‐ and 4.1‐fold (48, 72 h)), IGF‐IR (2‐fold vs. no‐significant reduction (72 h)) and IGFBP5 (3.24 vs. 1.38 (48 h) and 2.21 vs. 1.71 (72 h), P < 0.05) mRNA were observed in C2 versus C2C12 with TNF‐α. This investigation provides insight into regulators of altered basal hypertrophy and TNF‐induced atrophy, providing a model for future investigation into therapeutic initiatives for ageing/wasting disorders. J. Cell. Physiol. 225: 240–250, 2010.

Modelling in vivo skeletal muscle ageing in vitro using three-dimensional bioengineered constructs

Degeneration of skeletal muscle (SkM) with age (sarcopenia) is a major contributor to functional decline, morbidity and mortality. Methodological implications often make it difficult to embark on interventions in already frail and diseased elderly individuals. Using in vitro three‐dimensional (3D) bioengineered skeletal muscle constructs that model aged phenotypes and incorporate a representative extracellular matrix (collagen), are under tension, and display morphological and transcript expression of mature skeletal muscle may more accurately characterize the SkM niche. Furthermore, an in vitro model would provide greater experimental manipulation with regard to gene, pharmacological and exercise (mechanical stretch/electrical stimulation) therapies and thus strategies for combating muscle wasting with age. The present study utilized multiple population‐doubled (MPD) murine myoblasts compared with parental controls (CON), previously shown to have an aged phenotype in monolayer cultures ( Sharples et al., 2011 ), seeded into 3D type I collagen matrices under uniaxial tension. 3D bioengineered constructs incorporating MPD cells had reduced myotube size and diameter vs. CON constructs. MPD constructs were characterized by reduced peak force development over 24 h after cell seeding, reduced transcript expression of remodelling matrix metalloproteinases, MMP2 and MMP9, with reduced differentiation/hypertrophic potential shown by reduced IGF‐I, IGF‐IR, IGF‐IEa, MGF mRNA. Increased IGFBP2 and myostatin in MPD vs. CON constructs also suggested impaired differentiation/reduced regenerative potential. Overall, 3D bioengineered skeletal muscle constructs represent an in vitro model of the in vivo cell niche with MPD constructs displaying similar characteristics to ageing/atrophied muscle in vivo, thus potentially providing a future test bed for therapeutic interventions to contest muscle degeneration with age.

Reduction of myoblast differentiation following multiple population doublings in mouse C2C12 cells: A model to investigate ageing?

Ageing skeletal muscle displays declines in size, strength, and functional capacity. Given the acknowledged role that the systemic environment plays in reduced regeneration (Conboy et al. [2005] Nature 433: 760–764), the role of resident satellite cells (termed myoblasts upon activation) is relatively dismissed, where, multiple cellular divisions in‐vivo throughout the lifespan could also impact on muscular deterioration. Using a model of multiple population doublings (MPD) in‐vitro thus provided a system in which to investigate the direct impact of extensive cell duplications on muscle cell behavior. C2C12 mouse skeletal myoblasts (CON) were used fresh or following 58 population doublings (MPD). As a result of multiple divisions, reduced morphological and biochemical (creatine kinase, CK) differentiation were observed. Furthermore, MPD cells had significantly increased cells in the S and decreased cells in the G1 phases of the cell cycle versus CON, following serum withdrawal. These results suggest continued cycling rather than G1 exit and thus reduced differentiation (myotube atrophy) occurs in MPD muscle cells. These changes were underpinned by significant reductions in transcript expression of: IGF‐I and myogenic regulatory factors (myoD and myogenin) together with elevated IGFBP5. Signaling studies showed that decreased differentiation in MPD was associated with decreased phosphorylation of Akt, and with later increased phosphorylation of JNK1/2. Chemical inhibition of JNK1/2 (SP600125) in MPD cells increased IGF‐I expression (non‐significantly), however, did not enhance differentiation. This study provides a potential model and molecular mechanisms for deterioration in differentiation capacity in skeletal muscle cells as a consequence of multiple population doublings that would potentially contribute to the ageing process. J. Cell. Biochem. 112: 3773–3785, 2011.

Myoblast models of skeletal muscle hypertrophy and atrophy

Purpose of reviewTo highlight recent breakthroughs and controversies in the use of myoblast models to uncover cellular and molecular mechanisms regulating skeletal muscle hypertrophy and atrophy. Recent findingsMyoblast cultures provide key mechanistic models of the signalling and molecular pathways potentially employed by skeletal muscle in-vivo to regulate hypertrophy and atrophy. Recently the controversy as to whether insulin-like growth factor (IGF)-I is important in hypertrophy following mechanical stimuli vs. alternative pathways has been hotly debated and is discussed. The role of myostatin in myoblast models of atrophy and interactions between protein synthetic pathways including Akt/mTOR and the ‘atrogenes’ are explored. SummaryTargeted in-vivo experimentation directed by skeletal muscle cell culture and bioengineering (three-dimensional skeletal muscle cell culture models) will provide key biomimetic and mechanistic data regarding hypertrophy and atrophy and thus enable the development of important strategies for tackling muscle wasting associated with ageing and disease processes.

Postprandial Triacylglycerol in Adolescent Boys: A Case for Moderate Exercise

PURPOSE To compare the effects of 60-min bouts of intermittent moderate and vigorous exercise on postprandial plasma triacylglycerol (TAG) metabolism in eight healthy adolescent boys (mean +/- SD age: 13 +/- 0.3 yr). METHODS Participants completed three conditions in a counterbalanced order. On day 1, they either rested for 110 min (CON), completed 6 x 10-min blocks of intermittent treadmill exercise at 53% peak V O2 (MOD), or 6 x 10-min blocks at 75% peak V O2 (VIG). On day 2 after a 12-h fast, a capillary blood sample was taken for [TAG] and [glucose] (mmol.L) and then a high-fat milkshake was consumed (1.50 g.kg fat, 1.22 g.kg CHO, and 0.22 g.kg protein; 80 kJ.kg). Further blood samples were taken every hour for a 6-h postprandial rest period for [TAG] and [glucose]. RESULTS Estimated energy expenditure was 45% higher in VIG than in MOD (95% confidence interval [CI] 23-72%). Fasting [TAG] and [glucose] did not differ between the conditions. Average [TAG] for the postprandial period was lower by 24% in MOD (95% CI -47% to 9%, P = 0.06) and by 21% in VIG (95% CI -42% to 8%, P = 0.08) than CON, with no meaningful difference (4%; 95% CI -27% to 48%, P = 0.50) between MOD and VIG. The total area under the [TAG] versus time curve (mmol.L 6 h) was lower by 24% in MOD (95% CI -42% to 0%, P = 0.05) and by 20% in VIG (95% CI -37% to 0%, P = 0.07) than CON. MOD and VIG were not different from each other (4%; 95% CI -18% to 32%, P = 0.54). CONCLUSION Both 60 min of moderate and vigorous intermittent exercises reduced postprandial [TAG]. However, the extra energy expended in the vigorous condition did not produce a dose-related reduction compared with the moderate-intensity condition.

The role of insulin-like-growth factor binding protein 2 (IGFBP2) and phosphatase and tensin homologue (PTEN) in the regulation of myoblast differentiation and hypertrophy.

The complex actions of the insulin-like-growth factor binding proteins (IGFBPs) in skeletal muscle are becoming apparent, with IGFBP2 being implicated in skeletal muscle cell proliferation and differentiation (Ernst et al., 1992; Sharples et al., 2010). Furthermore, PTEN signalling has been linked to IGFBP2 action in other cell types by co-ordinating downstream Akt signalling, a known modulator of myoblast differentiation. The present study therefore aimed to determine the interaction between IGFBP2 and PTEN on myoblast differentiation. It has previously been established that C2C12 cells have high IGFBP2 gene expression upon transfer to low serum media, and that expression reduces rapidly as cells differentiate over 72 h [1]. Wishing to establish a potential role for IGFBP2 in this model, a neutralising IGFBP2 antibody was administered to C2C12 myoblasts upon initiation of differentiation. Myoblasts subsequently displayed reduced morphological differentiation (myotube number), biochemical differentiation (creatine kinase) and myotube hypertrophy (myotube area) with an early reduction in Akt phosphorylation. Knock-down of phosphatase and tensin homologue (PTEN) using siRNA in the absence of the neutralising antibody did not improve differentiation or hypertrophy vs. control conditions, however, in the presence of the neutralising IGFBP2 antibody, differentiation was restored and importantly hypertrophy exceeded that of control levels. Overall, these data suggest that; 1) reduced early availability of IGFBP2 can inhibit myoblast differentiation at later time points, 2) knock-down of PTEN levels can restore myoblast differentiation in the presence of neutralising IGFBP2 antibody, and 3) PTEN inhibition acts as a potent inducer of myotube hypertrophy when the availability of IGFBP2 is reduced in C2C12 myoblasts.

A Systems Based Investigation into Vitamin D and Skeletal Muscle Repair, Regeneration and Hypertrophy

Skeletal muscle is a direct target for vitamin D. Observational studies suggest that low 25[OH]D correlates with functional recovery of skeletal muscle following eccentric contractions in humans and crush injury in rats. However, a definitive association is yet to be established. To address this gap in knowledge in relation to damage repair, a randomised, placebo-controlled trial was performed in 20 males with insufficient concentrations of serum 25(OH)D (45 ± 25 nmol/l). Prior to and following 6 wk of supplemental vitamin D3 (4,000 IU/day) or placebo (50 mg of cellulose), participants performed 20 × 10 damaging eccentric contractions of the knee extensors, with peak torque measured over the following 7 days of recovery. Parallel experimentation using isolated human skeletal muscle-derived myoblast cells from biopsies of 14 males with low serum 25(OH)D (37 ± 11 nmol/l) were subjected to mechanical wound injury, which enabled corresponding in vitro studies of muscle repair, regeneration, and hypertrophy in the presence and absence of 10 or 100 nmol 1α,25(OH)2D3. Supplemental vitamin D3 increased serum 25(OH)D and improved recovery of peak torque at 48 h and 7 days postexercise. In vitro, 10 nmol 1α,25(OH)2D3 improved muscle cell migration dynamics and resulted in improved myotube fusion/differentiation at the biochemical, morphological, and molecular level together with increased myotube hypertrophy at 7 and 10 days postdamage. Together, these preliminary data are the first to characterize a role for vitamin D in human skeletal muscle regeneration and suggest that maintaining serum 25(OH)D may be beneficial for enhancing reparative processes and potentially for facilitating subsequent hypertrophy.

Sirtuin 1 regulates skeletal myoblast survival and enhances differentiation in the presence of resveratrol

Sirtuin 1 also known as NAD‐dependent deacetylase sirtuin 1, is a protein that in humans is encoded by the Sirt1 gene. Sirt1 is an enzyme that deacetylates proteins that contribute to cellular regulation and is a key regulator of cell defenses and survival in response to stress. Deletion of Sirt1 abolishes the increase in lifespan induced by calorie restriction or sublethal cytokine stress, indicating that Sirt1 promotes longevity and survival. We have demonstrated that administration of a sublethal dose of tumour necrosis factor‐α (TNF‐α; 1.25 ng ml−1) inhibits myotube formation, and co‐incubation with insulin‐like growth factor I (IGF‐I; 1.5 ng ml−1) facilitates C2 myoblast death rather than rescuing differentiation. A higher dose of TNF‐α (10 ng ml−1) resulted in significant apoptosis, which was rescued by IGF‐I (1.5 ng ml−1; 50% rescue; P < 0.05). We aimed to investigate the role of Sirt1 in the conflicting roles of IGF‐I. Quantitative real‐time PCR revealed that Sirt1 expression was elevated in myoblasts following incubation of 10 ng ml−1 TNF‐α or 1.25 ng ml−1 TNF‐α plus IGF‐I (fivefold and 7.2‐fold increases versus control, respectively; P < 0.05). A dose of 10 ng ml−1 TNF‐α induced ∼21 ± 0.7% apoptosis, which was reduced (∼50%; P < 0.05) when administered with IGF‐I. Likewise, Sirt1 expression was elevated following 10 ng ml−1 TNF‐α administration, but was reduced (∼30%; P < 0.05) in the presence of IGF‐I. C2C12 myoblasts, a subclone of the C2 cell line produced for their differentiation potential and used to examine intrinsic ageing, unlike C2 cells, do not die in the presence of TNF‐α and do not upregulate Sirt1. As conditions that induced the greatest myoblast stress/damage resulted in elevated Sirt1 expression, we investigated the effects of Sirt1 gene silencing. Treatment with 10 ng ml−1 TNF‐α or co‐incubation with 1.25 ng ml−1 TNF‐α and 1.5 ng ml−1 IGF‐I resulted in apoptosis (20.33 ± 2.08 and 19 ± 2.65%, respectively), which was increased when myoblasts were pretreated with Sirt1 small interfering RNA (31 ± 2.65 and 27.33 ± 2.52%, respectively; P < 0.05) and was reduced (14.33 ± 3.05%, P < 0.05 and 12.78 ± 4.52%, P= 0.054) by resveratrol, which also significantly rescued the block on differentiation. In conclusion, Sirt1 expression increases in conditons of stress, potentially serving to reduce or dampen myoblast death.

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.